Lytic domain fusion constructs and methods of making and using same

ABSTRACT

The invention relates to fusion constructs, methods of using fusion constructs and methods of treating undesirable or aberrant cell proliferation or hyperproliferative disorders, such as tumors, cancers, neoplasia and malignancies.

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 12/398,965, filed Mar. 5, 2009, now U.S. Pat. No. 8,546,535, whichis a continuation application of U.S. application Ser. No. 12/359,906,filed Jan. 26, 2009, now U.S. Pat. No. 8,318,899, which claims thebenefit of priority to U.S. Application Ser. No. 61/023,377, filed Jan.24, 2008, all of which applications are expressly incorporated herein byreference in their entirety.

TECHNICAL FIELD

The invention relates to fusion constructs, methods of using fusionconstructs and methods of treating undesirable or aberrant cellproliferation or hyperproliferative disorders, such as non-metastaticand metastatic neoplasias, cancers, tumors and malignancies.

INTRODUCTION

The need to develop new therapeutics for treatment of primary tumors andmetastases is clearly evident when the five year survival rate of cancerpatients is considered: Only 10-40% for patients with lung, colorectal,breast and prostate cancer survive if diagnosed with distant metastaticdisease.

SUMMARY

The invention is based, at least in part on lytic domains fused orconjugated to a binding moiety, referred to herein as fusion constructs.Contact of a cell with a lytic domain is believed to cause disruption ofthe cell membrane which results in cell death. The binding moietytargets cells for destruction by the lytic domain, including undesirableor aberrant proliferating cells or hyperproliferating cells, such asnon-metastatic and metastatic neoplasias, cancers, tumors andmalignancies. A number of non-metastatic and metastatic neoplastic,cancer, tumor and malignant cells overexpress receptors or ligands. Forexample, many non-metastatic and metastatic neoplasias, cancers, tumorsand malignancies, express receptors for hormones (for example,luteinizing hormone/chorionic gonadotropin (LH/CG), or luteinizinghormone releasing hormone (LHRH etc.), growth factors, cytokines,antibodies etc., that can be used as binding moiety of the fusionconstruct.

Fusion constructs can be designed to target any cell or cell populationthat expresses the binding site for the binding moiety. Binding moietiessuch as ligands, antibodies and fragments thereof, growth factors,cytokines, etc., can be linked to a lytic domain to provide targetedkilling of cells that express or contain receptors, antigens,antibodies, ligands etc. thereby reducing or inhibiting cellproliferation or growth.

Fusion constructs do not require cells to divide in order to kill thetarget cells. Furthermore, the fusion constructs are not likely to beimmunogenic because they can be made to be relatively small in size. Inaddition, the fusion constructs kill multi-drug resistant cells.

Moreover, the fusion constructs can have greater cytotoxic activity (lowIC₅₀) in terms of anti-cell proliferative activity or killing activityand low hemolytic activity (HA₅₀), such that the ratio of IC₅₀:HA₅₀(IC₅₀/HA₅₀) is lower than other compounds with such activities. Forexample, fusion constructs can have greater anti-cell proliferativeactivity than Phor21-βCG-ala, Phor21-GSGGS-βCG-ala,Phor21-ASAAS-βCG-ala, or Phor 14-βCG-ala, as ascertained by a lower IC₅₀value; have a lower IC₅₀/HA₅₀ ratio than Phor21-βCG-ala,Phor21-GSGGS-βCG-ala, Phor21-ASAAS-βCG-ala, or Phor 14-βCG-ala; or havean IC₅₀/HA₅₀ ratio of less than about 0.02, 0.01, or 0.005.

In accordance with the invention, there are provided fusion constructsthat include a first and a second domain. In one embodiment, a fusionconstruct includes or consists of a first domain consisting of a 12, 13,15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27 or 28 residue L- orD-amino acid sequence that includes a peptide sequence selected from forexample, KFAKFAKKFAKFAKK (SEQ. ID NO. 1), KFAKFAKKFAKFAKKF (SEQ. ID NO.2), KFAKFAKKFAKFAKKFA (SEQ. ID NO. 3), KFAKFAKKFAKFAKKFAK (SEQ. ID NO.4), KFAKFAKKFAKFAKKFAKF (SEQ. ID NO. 5), KFAKFAKKFAKFAKKFAKFA (SEQ. IDNO. 6) and KFAKFAKKFAKFAKKFAKFAKKFAKFAK (SEQ. ID NO. 7), and a seconddomain that includes or consists of a targeting or binding moiety. Inanother embodiment, a fusion construct includes or consists of a firstdomain consisting of an L- or D-amino acid sequence selected fromKFAKFAKKFAKFAKK (SEQ. ID NO. 1), KFAKFAKKFAKFAKKF (SEQ. ID NO. 2),KFAKFAKKFAKFAKKFA (SEQ. ID NO. 3), KFAKFAKKFAKFAKKFAK (SEQ. ID NO. 4),KFAKFAKKFAKFAKKFAKF (SEQ. ID NO. 5), KFAKFAKKFAKFAKKFAKFA (SEQ. ID NO.6) and KFAKFAKKFAKFAKKFAKFAKKFAKFAK (SEQ. ID NO. 7), and a second domainthat includes or consist of a targeting or binding moiety. In a furtherembodiment, a fusion construct includes or consists of a first domainconsisting of an L- or D-amino acid sequence selected from,KFAKFAKKFAKFAKK (SEQ. ID NO. 1), KFAKFAKKFAKFAKKF (SEQ. ID NO. 2),KFAKFAKKFAKFAKKFA (SEQ. ID NO. 3), KFAKFAKKFAKFAKKFAK (SEQ. ID NO. 4),KFAKFAKKFAKFAKKFAKF (SEQ. ID NO. 5), KFAKFAKKFAKFAKKFAKFA (SEQ. ID NO.6) and KFAKFAKKFAKFAKKFAKFAKKFAKFAK (SEQ. ID NO. 7), and a second domainconsisting of a 1-25 L- or D-amino acid sequence (e.g., targeting orbinding moiety) distinct from said first domain.

In accordance with the invention, there are also provided isolated andpurified peptides that include or consist of a first domain. In variousembodiments, an isolated or purified peptide is KFAKFAKKFAKFAKK (SEQ. IDNO. 1), KFAKFAKKFAKFAKKF (SEQ. ID NO. 2), KFAKFAKKFAKFAKKFA (SEQ. ID NO.3), KFAKFAKKFAKFAKKFAK (SEQ. ID NO. 4), KFAKFAKKFAKFAKKFAKF (SEQ. ID NO.5) or KFAKFAKKFAKFAKKFAKFA (SEQ. ID NO. 6). In additional embodiments,an isolated or purified peptide is KFAKFAKKFAKFAKK (SEQ. ID NO. 1),KFAKFAKKFAKFAKKF (SEQ. ID NO. 2), KFAKFAKKFAKFAKKFA (SEQ. ID NO. 3),KFAKFAKKFAKFAKKFAK (SEQ. ID NO. 4), KFAKFAKKFAKFAKKFAKF (SEQ. ID NO. 5)or KFAKFAKKFAKFAKKFAKFA (SEQ. ID NO. 6) having one or more of the Kresidues substituted with any of an F or L residue, one or more of the Fresidues substituted with any of a K, A or L residue, or one or more ofthe A residues substituted with any of a K, F or L residue.

Fusion constructs include a binding moiety that binds to a receptor,ligand, or an antigen. A binding moiety also includes a ligand, antigenor an antibody. Ligands include or consist of a molecule that binds to areceptor, such as a receptor agonist or antagonist. A binding moiety caninclude or consist of a linear or cyclic structure.

Specific non-limiting examples of binding moieties include one or moreamino acids (e.g., peptides, polypeptides, proteins), nucleic acids andcarbohydrates. Specific non-limiting classes of binding moieties includehormones, hormone analogues, and fragments of hormones and hormoneanalogs, growth factors, cytokines, antibodies etc. that bind to areceptor. Specific non-limiting examples of hormones include agonadotropin-releasing hormone or its analogs, luteinizing hormone betachain, luteinizing hormone, chorionic gonadotropin, chorionicgonadotropin beta subunit, melanocyte stimulating hormone, estradiol,diethylstilbesterol, lactoferrin, dopamine, somatostatin or its analogs,follicle-stimulating hormone (FSH), glucocorticoid, estrogen,testosterone, androstenedione, dihydrotestosterone,dehydroepiandrosterone, androgens, progesterone, thyroid stimulatinghormone (TSH), insulin, catecholamines, adrenocorticotropic hormone(ACTH), angiotensin, antidiuretic hormone, calcitonin, cholecystokinin,bombesin, corticotrophin-releasing hormone, gastrin, ghrelin, glucagon,Growth Hormone Releasing Hormone and its analogs, inhibin, orexin, KISSpeptide (GPR54), kisspeptin, Prolactin, Prolactin Releasing Hormone,Growth Hormone, Her2/neu, folate, vitamin H, ferritin, ParathyroidHormone, Relaxin, Secretin, Thyrotropin Releasing Hormone, Endothelin,Renin, Lipotropin, melatonin etc. Specific non-limiting examples ofgrowth factors are epidermal growth factor (EGF), insulin-like growthfactor-1 and 2 (IGF-1, IGF-2), vascular endothelial growth factor(VEGF), Nerve Growth Factor (NGF), Fibroblast Growth Factor (FGF),Transforming Growth Factor alpha and beta (TGFα, TGFβ, Platelet DerivedGrowth Factor (PDGF), Hepatocyte Growth Factor (HGF), ceruloplasmin etc.Specific non-limiting classes of cytokines or ligands are interleukins(for example interleukin 2, interleukin 17, CD154, Fas Ligand etc),Tumor Necrosis Factors (TNFs), interferons, etc.

Binding moieties can be optionally expressed on a cell. Cells thatexpress a binding moiety (e.g., receptor, ligand, antigen, antibody) orthat can be targeted in accordance with methods of the invention includehyperproliferative cells. Cells that express a receptor, ligand, orantigen, or that can be targeted in accordance with methods of theinvention also include breast, ovarian, uterine, cervical, prostate,testicular, pancreatic, skin, blood cells, adrenal, pituitary andendometrial cells. Specific non-limiting classes of binding moietiesexpressed on a cell are receptors for hormones, cytokines, growthfactors (for example EGF receptors, Her2/neu, ROR1), ferritin,transferrin receptors, cell adhesion molecules, etc. Specificnon-limiting examples of antigens expressed on proliferating cells thatcan be targeted with antibodies or their fragments are CD19, CD20, CD23,CD27, CD28, CD30, CD33, CD40, CD52, CD56, CD70, CD154,immunoglobulin-like receptors etc). Further antigens, include, forexample, prostate specific antigen (PSA), prostate specific membraneantigen (PSMA), carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP),prostate specific antigen (PSA), cancer antigen 125 (CA-125) and otherreceptor molecules that bind to ligands disclosed herein.

First and second domains can include or consist of an amino acid, or anamino acid sequence. In particular aspects, a first or second domain hasabout 1 to 10, 10 to 20, 15 to 20 (i.e., 15, 16, 17, 18, 19 or 20 aminoacids), 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70 to 80, 80 to 90, 90to 100 or more amino acid residues.

In a particular aspect, a first domain includes or consists of anamphipathic alpha-helical structure. In further particular aspects, asecond domain includes or consists of an amino acid sequence set forthas SYAVALSAQAALARR (SEQ. ID NO. 8) or QHWSYGLRPG (SEQ. ID NO. 9).

First and second domains can be positioned at either the NH-terminus orthe C-terminus relative to each other. Thus, in one embodiment the first(lytic peptide) domain is positioned at the NH-terminus relative to thesecond (binding moiety or ligand) domain, and in another embodiment, thesecond (binding moiety or ligand) domain is positioned at the C-terminusrelative to the first (lytic peptide) domain.

First and second domains can include or consist of one or more D-aminoacids. In particular aspects, a first domain has a D-amino acid, forexample, at any K, F or A residue.

First and second domains can be joined by a covalent bond. For example,a first and a second domain can be joined by a peptide or a non-peptidelinker. In particular aspects, first and second domains are joined by apeptide sequence having from about 1 to 25 amino acid residues, orhaving a linear carbon chain. In more particular aspects, first andsecond domains are joined by a peptide sequence that includes or consistof one or more A, S or G amino acid residues. In further particularaspects, first and second domains are joined by a peptide sequence afirst and second domain is joined by peptide sequence including orconsisting of GSGGS (SEQ. ID NO. 10), ASAAS (SEQ. ID NO. 11), or CCCCCC(SEQ. ID NO. 12).

First and second domains can further include or consist of additionaldomains. Thus, in various aspects, a fusion construct includes a third,fourth, fifth, sixth, seventh domain, etc.

Fusion constructs include or consist of isolated and purified forms.Fusion constructs also include or consist of a mixture. Such mixturesinclude compositions, such as a mixture of fusion construct and apharmaceutically acceptable carrier or excipient appropriate foradministration to or in vivo contact with a subject, or a mixture offusion construct and an anti-cell proliferative or immune stimulatingagent.

Fusion constructs include or consist of a unit dosage form. In oneembodiment, a fusion construct is a unit dosage in an amount effectiveto treat a subject having undesirable cell proliferation or ahyperproliferative disorder. In another embodiment, a fusion constructis a unit dosage in an amount effective to treat a subject having aneoplasia, tumor or cancer. In an additional embodiment, a fusionconstruct is a unit dosage in an amount effective to reduce fertility ofa subject.

Fusion constructs can be included within kits, optionally withinstructions for practicing a method. In one embodiment, a kit includesa fusion construct and instructions for reducing or inhibitingproliferation of a cell, reducing or inhibiting proliferation of ahyperproliferating cell, reducing or inhibiting proliferation of aneoplastic, tumor or cancer cell, treating a subject having ahyperproliferative disorder, treating a subject having a neoplasia,tumor or cancer, or reducing fertility of an animal.

In accordance with the invention, there are also provided nucleic acidsthat encodes fusion constructs. In one embodiment, a nucleic acidencodes a fusion construct including or consisting of a first domainconsisting of a 12, 13, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27or 28 residue L- or D-amino acid sequence that includes a peptidesequence selected from KFAKFAKKFAKFAKK (SEQ. ID NO. 1), KFAKFAKKFAKFAKKF(SEQ. ID NO. 2), KFAKFAKKFAKFAKKFA (SEQ. ID NO. 3), KFAKFAKKFAKFAKKFAK(SEQ. ID NO. 4), KFAKFAKKFAKFAKKFAKF (SEQ. ID NO. 5),KFAKFAKKFAKFAKKFAKFA (SEQ. ID NO. 6) and KFAKFAKKFAKFAKKFAKFAKKFAKFAK(SEQ. ID NO. 7), and a second domain including or consists of atargeting or binding moiety. In another embodiment, a nucleic acidencodes a fusion construct including or consisting of a first domainconsisting of an L- or D-amino acid sequence selected fromKFAKFAKKFAKFAKK (SEQ. ID NO. 1), KFAKFAKKFAKFAKKF (SEQ. ID NO. 2),KFAKFAKKFAKFAKKFA (SEQ. ID NO. 3), KFAKFAKKFAKFAKKFAK (SEQ. ID NO. 4),KFAKFAKKFAKFAKKFAKF (SEQ. ID NO. 5), KFAKFAKKFAKFAKKFAKFA (SEQ. ID NO.6) and KFAKFAKKFAKFAKKFAKFAKKFAKFAK (SEQ. ID NO. 7), and a second domainthat includes or consist of a targeting or binding moiety. In a furtherembodiment, a nucleic acid encodes a fusion construct including a firstdomain consisting of an L- or D-amino acid sequence selected fromKFAKFAKKFAKFAKK (SEQ. ID NO. 1), KFAKFAKKFAKFAKKF (SEQ. ID NO. 2),KFAKFAKKFAKFAKKFA (SEQ. ID NO. 3), KFAKFAKKFAKFAKKFAK (SEQ. ID NO. 4),KFAKFAKKFAKFAKKFAKF (SEQ. ID NO. 5), KFAKFAKKFAKFAKKFAKFA (SEQ. ID NO.6) and KFAKFAKKFAKFAKKFAKFAKKFAKFAK (SEQ. ID NO. 7), and a second domainincluding or consisting of a 1-25 L- or D-amino acid sequence (e.g.,targeting or binding moiety) distinct from said first domain.

Nucleic acids can be included in a vector, such as an expression vectorthat when expressed in a cell encodes a fusion construct. Host cells canbe transformed with a nucleic acid in a vector, such that the cellexpresses a fusion construct encoded by the nucleic acid.

Fusion constructs are useful for, among other things, reducing orinhibiting proliferation of a cell, reducing or inhibiting cellproliferation, reducing or inhibiting proliferation of ahyperproliferating cell, reducing or inhibiting proliferation of aneoplastic, tumor, cancer or malignant cell and treating undesirable oraberrant cell proliferation, such as hyperproliferating cells orhyperproliferative disorders. Non-limiting examples ofhyperproliferative disorders include benign hyperplasia, non-metastaticand metastatic neoplasias, cancers tumors and malignancies (e.g., asolid or liquid tumor, myeloma, lymphoma, leukemia, carcinoma, sarcoma,melanoma, neural, reticuloendothelial and haematopoietic).

In accordance with the invention, there are further provided methods ofreducing or inhibiting proliferation of a cell; methods of reducing orinhibiting cell proliferation; methods of reducing or inhibitingproliferation of a hyperproliferating cell; and methods of reducing orinhibiting proliferation of a neoplastic, tumor, cancer or malignantcell. In various embodiments, a method includes contacting a cell with afusion construct in an amount sufficient to reduce or inhibitproliferation of the cell; contacting a cell with a fusion construct inan amount sufficient to reduce or inhibit cell proliferation; contactinga cell with a fusion construct in an amount sufficient to reduce orinhibit proliferation of the hyperproliferating cell; and contacting acell with a fusion construct in an amount sufficient to reduce orinhibit proliferation of the neoplastic, tumor, cancer or malignantcell.

In accordance with the invention, there are moreover provided methods ofselectively reducing or inhibiting proliferation of a cell thatexpresses a receptor or antigen; selectively reducing or inhibitingproliferation of a hyperproliferating cell that expresses a receptor orantigen; and selectively reducing or inhibiting proliferation of aneoplastic, tumor, cancer or malignant cell that expresses a receptor orantigen. In various embodiments, a method includes contacting a cellwith the fusion construct in an amount sufficient to reduce or inhibitproliferation of the cell, wherein the binding moiety of said peptidebinds to the receptor, ligand, or antigen expressed by the cell;contacting a cell with the fusion construct in an amount sufficient toreduce or inhibit proliferation of the hyperproliferating cell, whereinthe binding moiety of said peptide binds to the receptor, ligand, orantigen expressed by the hyperproliferating cell; and contacting a cellwith the fusion construct in an amount sufficient to reduce or inhibitproliferation of the neoplastic, tumor, cancer or malignant cell,wherein the binding moiety of said fusion construct binds to thereceptor, ligand, or antigen expressed by the cell.

Cells targeted in accordance with the invention methods include cellsthat express a receptor, or an antigen, such as a hormone receptor, forexample, a sex or gonadal steroid hormone or a sex or gonadal steroidhormone receptor. Cells targeted in accordance with the inventionmethods also include cells that express a receptor that binds togonadotropin-releasing hormone I, gonadotropin-releasing hormone II,lamprey III luteinizing hormone releasing hormone, luteinizing hormonebeta chain, luteinizing hormone, chorionic gonadotropin, chorionicgonadotropin beta subunit, melanocyte stimulating hormone, estradiol,diethylstilbesterol, dopamine, somatostatin, follicle-stimulatinghormone (FSH), glucocorticoid, estrogen, testosterone, androstenedione,dihydrotestosterone, dehydroepiandrosterone, progesterone, androgen,prolactin, prolactin releasing hormone, antidiuretic hormone,angiotensins, catecholamines, epidermal growth factor (EGF), insulinlike growth factor-1 and 2 (IGF-1, IGF-2), growth hormone (GH),Her2/neu, vitamin H, folate, transferrin, thyroid stimulating hormone(TSH), parathyroid hormone (PTH), endothelin, bombesin, Renin,Lipotropin, melatonin hormone, relaxin, secretin, growth hormone,vascular endothelial growth factor (VEGF), vasoactive intestinalpeptide, lactoferrin, an integrin (e.g., alpha-5 beta 3 or alpha-5 beta1 integrin), nerve growth factor, transforming growth factor alpha andbeta (TGF-α and β), hepatocyte growth factor (HGF), fibroblast growthfactor (FGF), CD-33, CD19, CD20, CD40, ROR1, IGF-1, carcinoembryonicantigen (CEA), alpha-fetoprotein (AFP), prostate specific antigen (PSA),prostate specific membrane antigen (PSMA), cancer antigen 125 (CA-125),interleukin 17, CD154, soluble Interleukin-2 (IL-2) receptor,tyrosinase, MAGE-1, MAGE-2, NY-ESO-1, Melan-A/MART-1, glycoprotein (gp)75, gp100, beta-catenin, PRAME, MUM-1, ZFP161, Ubiquitin-1, HOX-B6,YB-1, Osteonectin, ILF3, folic acid or a derivative thereof, a tumornecrosis factor (TNF) family member, TNF-alpha, TNF-beta (lymphtoxin,LT), TRAIL, Fas, LIGHT, 41BB, transforming growth factor alpha,transforming growth factor beta, insulin, ceruloplasmin, HIV-tat, apeptide or protein comprising an RGD sequence motif, a mono-saccharide,di-saccharide, oligo-saccharide, sialic acid, galactose, mannose,fucose, or acetylneuraminic acid.

Methods performed include, among others, contacting a subject in need ofinhibiting, reducing or preventing proliferation, survival,differentiation, death, or activity of a cells, such as ahyperprolifertive cell or an undesirably proliferating cell. Exemplarysubjects include a subject having or at risk of having undesirable oraberrant cell proliferation; a subject having or at risk of having abenign hyperplasia; or a non-metastatic or metastatic neoplasia, cancer,tumor or malignancy (e.g., a solid or liquid tumor, myeloma, lymphoma,leukemia, carcinoma, sarcoma, melanoma, neural, reticuloendothelial andhaematopoietic neoplasia).

In accordance with the invention, there are additionally providedmethods of treating a subject having a hyperproliferative disorder andmethods of treating a subject having a neoplasia, tumor, cancer ormalignancy (metastatic, non-metastatic or benign). In variousembodiments, a method includes, administering to a subject an amount ofthe fusion construct sufficient to treat the hyperproliferativedisorder; and administering to a subject an amount of the fusionconstruct sufficient to reduce or inhibit proliferation of theneoplasia, tumor, cancer or malignancy.

Methods include treating a subject having or at risk of having ametastasis. For example, an amount of a fusion construct effective toreduce or inhibit spread or dissemination of a tumor, cancer orneoplasia to other sites, locations or regions within the subject. Invarious embodiments, a method reduces or inhibits metastasis of aprimary tumor or cancer to one or more other sites, formation orestablishment of a metastasis at one or more other sites, locations orregions thereby reducing or inhibiting tumor or cancer relapse or tumoror cancer progression. In further embodiments, a method reduces orinhibits growth, proliferation, mobility or invasiveness of tumor orcancer cells that potentially or do develop metastases (e.g.,disseminated tumor cells); reduces or inhibits formation orestablishment of metastases arising from a primary tumor or cancer toone or more other sites, locations or regions distinct from the primarytumor or cancer; reduces or inhibits growth or proliferation of ametastasis at one or more other sites, locations or regions distinctfrom the primary tumor or cancer after the metastasis has formed or hasbeen established; or reduces or inhibits formation or establishment ofadditional metastasis after the metastasis has been formed orestablished. In yet another embodiment, a method reduces or inhibitsrelapse or progression of the neoplasia, tumor, cancer or malignancy.

In accordance with the invention, there are still further providedmethods of reducing or inhibiting metastasis of a neoplasia, tumor,cancer or malignancy to other sites, or formation or establishment ofmetastatic neoplasia, tumor, cancer or malignancy at other sites distalfrom a primary neoplasia, tumor, cancer or malignancy. In variousembodiments, a method includes administering to a subject an amount ofthe fusion construct sufficient to reduce or inhibit metastasis of theneoplasia, tumor, cancer or malignancy to other sites, or formation orestablishment of metastatic neoplasia, tumor, cancer or malignancy atother sites distal from the primary neoplasia, tumor, cancer ormalignancy.

Neoplasia, tumor, cancer and malignancy treatable in accordance with theinvention include solid cellular mass, hematopoietic cells, or acarcinoma, sarcoma (e.g. lymphosarcoma, liposarcoma, osteosarcoma,chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma or fibrosarcoma),lymphoma, leukemia, adenoma, adenocarcinoma, melanoma, glioma,glioblastoma, meningioma, neuroblastoma, retinoblastoma, astrocytoma,oligodendrocytoma, mesothelioma, reticuloendothelial, lymphatic orhaematopoietic (e.g., myeloma, lymphoma or leukemia) neoplasia, tumor,cancer or malignancy.

Neoplasia, tumor, cancer and malignancy treatable in accordance with theinvention can be present in or affect a lung (small cell lung ornon-small cell lung cancer), thyroid, head or neck, nasopharynx, throat,nose or sinuses, brain, spine, breast, adrenal gland, pituitary gland,thyroid, lymph, gastrointestinal (mouth, esophagus, stomach, duodenum,ileum, jejunum (small intestine), colon, rectum), genito-urinary tract(uterus, ovary, cervix, endometrial, bladder, testicle, penis,prostate), kidney, pancreas, liver, bone, bone marrow, lymph, blood,muscle, skin or stem cell neoplasia, tumor, cancer, or malignancy.

Methods may be practiced with other treatments or therapies (e.g.,surgical resection, radiotherapy, ionizing or chemical radiationtherapy, chemotherapy, immunotherapy, local or regional thermal(hyperthermia) therapy, or vaccination). Such treatments or therapiescan be administered prior to, substantially contemporaneously with(separately or in a mixture), or following administration of a fusionconstruct. In one embodiment, a method includes administering ananti-cell proliferative, anti-neoplastic, anti-tumor, anti-cancer orimmune-enhancing treatment or therapy. In further embodiments, a methodincludes administering an alkylating agent, anti-metabolite, plantextract, plant alkaloid, nitrosourea, hormone, nucleoside or nucleotideanalog; cyclophosphamide, azathioprine, cyclosporin A, prednisolone,melphalan, chlorambucil, mechlorethamine, busulphan, methotrexate,6-mercaptopurine, thioguanine, 5-fluorouracil, cytosine arabinoside,5-azacytidine (5-AZC) and 5-azacytidine related compounds, bleomycin,actinomycin D, mithramycin, mitomycin C, carmustine, lomustine,semustine, streptozotocin, hydroxyurea, cisplatin, carboplatin,oxiplatin, mitotane, procarbazine, dacarbazine, taxol, vinblastine,vincristine, doxorubicin or dibromomannitol, topoisomerase inhibitors,(irinotecan, topotecan, etoposide, teniposide), gemcitabine, pemetrexedetc. Cell or immunotherapies include a lymphocytes, plasma cells,macrophages, dendritic cells, T-cells, NK cells or B-cells; an antibody,a cell growth factor, a cell survival factor, a cell differentiativefactor, a cytokine or a chemokine (examples are interleukins IL-2,IL-1α, IL-13, IL-3, IL-6, IL-7, granulocyte-macrophage-colonystimulating factor (GMCSF), IFN-γ, IL-12, TNF-α, TNFβ, MIP-1α, MIP-1β,RANTES, SDF-1, MCP-1, MCP-2, MCP-3, MCP-4, eotaxin, eotaxin-2,I-309/TCA3, ATAC, HCC-1, HCC-2, HCC-3, LARC/MIP-3α, PARC, TARC, CKβ,CKβ6, CKβ7, CKβ8, CKβ9, CKβ11, CKβ12, C10, IL-8, GROα, GROβ, ENA-78,GCP-2, PBP/CTAPIIIβ-TG/NAP-2, Mig, PBSF/SDF-1, or lymphotactin) etc.

Additional agents that are applicable with fusion constructs aretargeted drugs or biological such as antibodies or small molecules.Non-limiting examples of monoclonal antibodies include rituximab(Rituxan®), trastuzumab (Herceptin), bevacizumab (Avastin), cetuximab(Erbitux), alemtuzumab (Campath), panitumumab (Vectibix), ibritumomabtiuxetan (Zevalin), tositumomab (Bexxar) etc. which can be used incombination with, inter alia, a fusion construct in accordance with theinvention. Other targeted drugs that are applicable for use with thefusion constructs are imatinib (Gleevec), gefitinib (Iressa), bortzomib(Velcade), lapatinib (Tykerb), sunitinib (Sutent), sorafenib (Nevaxar),nilotinib (Tasigna) etc.

Methods of the invention include providing a subject with a benefit. Inparticular embodiments, a method of treatment results in partial orcomplete destruction of the neoplastic, tumor, cancer or malignant cellmass, volume, size or numbers of cells, stimulating, inducing orincreasing neoplastic, tumor, cancer or malignant cell necrosis, lysisor apoptosis, reducing neoplasia, tumor, cancer or malignancy volumesize, cell mass, inhibiting or preventing progression or an increase inneoplasia, tumor, cancer or malignancy volume, mass, size or cellnumbers, or prolonging lifespan; results in reducing or decreasingseverity, duration or frequency of an adverse symptom or complicationassociated with or caused by the neoplasia, tumor, cancer or malignancy;results in reducing or decreasing pain, discomfort, nausea, weakness orlethargy; or results in increased energy, appetite, improved mobility orpsychological well being.

In accordance with the invention, there are still additionally providedmethods of reducing fertility of an animal; methods treating or reducingendometriosis, benign prostate hyperplasia, a fibroid or polyp. Invarious embodiments, a method includes administering to an animal (e.g.,mammal, such as a human) an amount of a fusion construct sufficient toreduce fertility; administering to an animal (e.g., mammal, such as ahuman) an amount of a fusion construct sufficient to treat or reduceendometriosis; administering to an animal (e.g., mammal, such as ahuman) an amount of a fusion construct sufficient to treat or reducebenign prostate hyperplasia; and administering to an animal (e.g.,mammal, such as a human) an amount of a fusion construct sufficient totreat or reduce a fibroid or polyp.

Subjects treatable in accordance with the methods include mammals. Inparticular embodiments, a subject is a human.

DESCRIPTION OF DRAWINGS

FIG. 1 shows that LHRH-Phor21 kills cancer cells faster thanPhor21-βCG-ala. Human breast cancer cells (MDA-MB-435S.luc, variouspassage numbers) were incubated with Phor21-βCG-ala or LHRH-Phor21.

FIG. 2 shows cytotoxicity to MDA-MB-435S.luc cell (micromolar IC₅₀) ofβCG-ala fusion constructs having 21 (Phor21), 18 (Phor18(338983)=CLIP71) and 15 (Phor15) amino acids in their lytic domain,compared to Phor21-βCG-ala.

FIG. 3 shows cytotoxicity to MDA-MB-435S.luc cells (micromolar IC₅₀) ofβCG-ala and LHRH fusion constructs. Fusion constructs more toxic toMDA-MB-435S.luc cells than Phor21-βCG-ala are listed to the right of thefigure.

FIG. 4 shows cytotoxicity to MDA-MB-435S.luc cell (micromolar IC₅₀) ofLHRH fusion constructs. The fusion constructs are:323033=Phor21-βCG-ala, 337479=LHRH-Phor21, 337480=Phor21-LHRH,338611=D-ala-Phor21l-LHRH, 338612=Phor18-ASAAS-LHRH, 338613=Phor18-LHRH,339385=D-ala-Phor18-LHRH, and 339347=Phor18-Lupron.

FIG. 5 shows acute hemolytic activity (micromolar HA₅₀) of βCG-ala andLHRH fusion constructs to human red blood cells compared toPhor21-βCG-ala. All fusion constructs had significantly lower hemolyticactivity than Phor21-βCG-ala, except for LHRH-Phor21, Phor21-LHRH andPhor18-Lupron (QHWSY(D-Leu)LRPNEt=Lupron).

FIG. 6 shows a comparison of cytotoxicity and hemolytic activity.Peptides indicated with arrows are more toxic to cells thanPhor21-βCG-ala.

FIG. 7 is a treatment schedule outline.

FIGS. 8A-8J is a summary of tumor conditions during treatment and atstudy endpoint with 3 βCG conjugates in comparison to unconjugatedPhor21, unconjugated Phor18 (338983)=(CLIP71) and an (KKKFAFA)₃ (SEQ. IDNO. 16) conjugate (338984). Fusion construct codes, 33=Phor21β-CG-ala;76=Phor18-βCG-ala; 81=D-ala-Phor21-βCG-ala; 85=D-ala-Phor18-LHRH;47=Phor18-Lupron; 13=Phor18-LHRH; 11=D-ala-Phor21-LHRH;12=Phor18-ASAAS-LHRH; 71=Phor15-βCG-ala; and 74=Phor15-C6-βCG-ala arefollowed by amounts of the construct used in the study.

FIGS. 9A-9H show tumor volume of treatment groups compared to saline andbaseline values for A) Phor21-βCG-ala (33); B) D-ala-Phor21-LHRH (11);C) Phor18-Lupron (47); D) Phor18-ASAAS-LHRH (12); E) Phor18-LHRH (13);F) (KKKFAFA)₃ (SEQ. ID NO. 16)-LHRH; G) D-ala-Phor18-LHRH (85) at theindicated time periods up to 30 days; and H) compared to baseline.

FIGS. 10A-10E show a summary of tumor conditions at study endpoint with5 LHRH conjugates in comparison to Phor21-βCG-ala for A) Tumor weights,B) Tumor weight change compared to baseline, C) Total number of livetumor cells, D) changes of total number of live tumor cells compared tobaseline, and E) bodyweights. 338614=(KKKFAFA)₃ (SEQ. ID NO. 16) LHRH,338612=Phor18-ASAAS-LHRH, 338613=Phor18-LHRH, and339385=D-ala-Phor18-LHRH.

FIG. 11 shows ovarian cancer cells (OVCAR 3), which are multi-drugresistant, incubated with increasing concentrations of Phor21-βCG-ala inthe presence of Doxorubicin at the indicated amounts, and potentiationof cell killing by a factor of 200 at the highest doxorubicinconcentration by the combination.

FIG. 12 shows CHO (Chinese Hamster Ovary) and TM4 cell cytotoxicity incomparison to MDA-MB-435S.luc cells with LHRH and Phor21 and βCG andPhor21 fusion constructs. TM4 cells are LHRH receptor negative, CHOcells are CG receptor negative, and MDA-MB-435S.luc cells express bothLHRH and CG receptors.

DETAILED DESCRIPTION

The invention is based at least in part on a fusion construct thatincludes a first domain lytic portion joined or fused to a second domainbinding portion. In a typical configuration, a fusion construct firstdomain includes a lytic portion, which is directly or indirectly toxicto a cell, which can thereby reduce cell proliferation or survival, orstimulate, induce, increase or enhance cell death, killing or apoptosis;and a fusion construct second domain includes a portion that targets acell, referred to as a binding moiety entity.

In accordance with the invention, there are provided fusion constructsthat include or consist of a first “lytic” domain and include or consistof a second “targeting” or “binding” domain. In one embodiment, a fusionconstruct includes a first domain consisting of a 12, 13, 15, 16, 17,18, 19, 20, 22, 23, 24, 25, 26, 27 or 28 residue L- or D-amino acidsequence that includes a peptide sequence (selected from amino acidssuch as Lysine=K, Phenylalanine=F and Alanine=A), for example,KFAKFAKKFAKFAKK (SEQ. ID NO. 1), KFAKFAKKFAKFAKKF (SEQ. ID NO. 2),KFAKFAKKFAKFAKKFA (SEQ. ID NO. 3), KFAKFAKKFAKFAKKFAK (SEQ. ID NO. 4),KFAKFAKKFAKFAKKFAKF (SEQ. ID NO. 5), KFAKFAKKFAKFAKKFAKFA (SEQ. ID NO.6) and KFAKFAKKFAKFAKKFAKFAKKFAKFAK (SEQ. ID NO. 7), and a second domainthat includes or consists of a targeting or binding moiety. In anotherembodiment, a fusion construct includes a first domain consisting of anL- or D-amino acid sequence selected from KFAKFAKKFAKFAKK (SEQ. ID NO.1), KFAKFAKKFAKFAKKF (SEQ. ID NO. 2), KFAKFAKKFAKFAKKFA (SEQ. ID NO. 3),KFAKFAKKFAKFAKKFAK (SEQ. ID NO. 4), KFAKFAKKFAKFAKKFAKF (SEQ. ID NO. 5),KFAKFAKKFAKFAKKFAKFA (SEQ. ID NO. 6) and KFAKFAKKFAKFAKKFAKFAKKFAKFAK(SEQ. ID NO. 7), and a second domain that includes or consist of atargeting or binding moiety. In a further embodiment, a fusion constructincludes or consists of a first domain consisting of an L- or D-aminoacid sequence selected from KFAKFAKKFAKFAKK (SEQ. ID NO. 1),KFAKFAKKFAKFAKKF (SEQ. ID NO. 2), KFAKFAKKFAKFAKKFA (SEQ. ID NO. 3),KFAKFAKKFAKFAKKFAK (SEQ. ID NO. 4), KFAKFAKKFAKFAKKFAKF (SEQ. ID NO. 5),KFAKFAKKFAKFAKKFAKFA (SEQ. ID NO. 6) and KFAKFAKKFAKFAKKFAKFAKKFAKFAK(SEQ. ID NO. 7), and a second domain consisting of a 1-25 L- or D-aminoacid sequence (e.g., targeting or binding moiety) distinct from saidfirst domain.

As used herein, the term “fusion” or “chimeric” and grammaticalvariations thereof, when used in reference to a construct, means thatthe construct contains portions or sections that are derived from,obtained or isolated from, or are based upon or modeled after twodifferent molecular entities that are distinct from each other and donot typically exist together in nature. That is, for example, oneportion of the fusion construct includes or consists of a lytic portionand a second portion of the construct includes or consists of atargeting portion, such as a moiety that has binding capability, each offirst and second domains structurally distinct. A fusion construct canalso be referred to as a “conjugate,” wherein the conjugate includes orconsists of a first domain lytic portion and a second domain targetingor binding moiety.

First domains and or second domains of fusion constructs include orconsist of amino acid sequences (peptides, polypeptides, proteins,lectins), nucleic acids (DNA, RNA) and carbohydrates (saccharides,sialic acid, galactose, mannose, fucose, acetylneuraminic acid, etc.).The terms “amino acid sequence,” “protein,” “polypeptide” and “peptide”are used interchangeably herein to refer to two or more amino acids, or“residues,” covalently linked by an amide bond or equivalent. Amino acidsequences can be linked by non-natural and non-amide chemical bondsincluding, for example, those formed with glutaraldehyde,N-hydroxysuccinimide esters, bifunctional maleimides, orN,N′-dicyclohexylcarbodiimide (DCC). Non-amide bonds include, forexample, ketomethylene, aminomethylene, olefin, ether, thioether and thelike (see, e.g., Spatola in Chemistry and Biochemistry of Amino Acids,Peptides and Proteins, Vol. 7, pp 267-357 (1983), “Peptide and BackboneModifications,” Marcel Decker, NY).

First and second domains of a fusion construct or chimera includeL-amino acid sequences, D-amino acid sequences and amino acid sequenceswith mixtures of L-amino acids and D-amino acids. Amino acid sequencesof first and second domains can be a linear or a cyclic structure,conjugated to a distinct moiety (e.g., third, fourth, fifth, sixth,seventh, etc. domains), form intra or intermolecular disulfide bonds,and also form higher order multimers or oligomers with the same ordifferent amino acid sequence, or other molecules.

Exemplary lengths of fusion constructs are from about 5 to 15, 20 to 25,25 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 300 or more aminoacid residues in length. In particular embodiments, a first or seconddomain includes or consists of an amino acid sequence of about 1 to 10,10 to 20, 15 to 20, 20 to 30, 30 to 40, 40 to 50, 60 to 70, 70 to 80, 80to 90, 90 to 100 or more residues. In more particular embodiments, afirst domain consists of a 15, 16, 17, 18, 19, 20, 28 or more residueamino acid sequence.

Fusion construct first domains, alone or in combination with a seconddomain, optionally form an amphipathic alpha-helix. An amphipathicalpha-helix contains mostly hydrophilic amino acids on one side of thealpha-helix and the other side contains mostly hydrophobic amino acids.Since the alpha helix makes a complete turn for every 3.6 residues, theamino acid sequence of an amphipathic alpha helix alternates betweenhydrophilic and hydrophobic residues every 3 to 4 residues. A PNNPNNP(SEQ. ID NO. 14) repeat pattern or motif is predicted to form anamphipathic alpha-helix where P represents a positively charged aminoacid residue and N a neutral amino acid residue. A PNNPNNP (SEQ. ID NO.14) repeat pattern provides a cationic binding site for the lyticpeptide to a negatively charged cell membrane and a hydrophobic site formembrane interaction/penetration. Fusion constructs therefore includefirst domains with one or more uninterrupted PNNPNNP (SEQ. ID NO. 14)repeat patterns or motifs, or one or more interrupted PNNPNNP (SEQ. IDNO. 14) repeat patterns or motifs, which can form an amphipathicalpha-helix. For example, a 15 or 18 residue amino acid sequence, suchas KFAKFAKKFAKFAKK (SEQ. ID NO. 1) and KFAKFAKKFAKFAKKFAK (SEQ. ID NO.4), has uninterrupted and interrupted PNNPNNP (SEQ. ID NO. 14) repeatmotifs.

A fusion construct second domain, such as a targeting or binding moiety,includes or consists of a ligand, antibody (or an antigen-bindingfragment thereof), antigen, integrin, integrin receptor (e.g., proteinsor peptides containing “RGD” sequence motif, and components that may bepresent in extracellular matrix (ECM), such as mono-, di- oroligo-saccharides, sialic acid, galactose, mannose, fucose,acetylneuraminic acid), growth factor, cytokine, chemokine, andtargeting and binding moieties that bind to receptors, antibodies,antigens, integrins, integrin receptors (e.g., proteins or peptidescontaining “RGD” sequence motif, and components that may be present inextracellular matrix (ECM), such as mono-, di- or oligo-saccharides,sialic acid, galactose, mannose, fucose, acetylneuraminic acid), growthfactor receptors, cytokine receptors, and chemokine receptors.

A “receptor” is typically present on (e.g., a membrane receptor) orwithin a cell. A receptor may associate with the cell membrane surfaceor traverse the cell membrane. For example, a receptor protein can havea transmembrane domain that traverses the cell membrane, optionally witha portion that is cytoplasmic or extracellular, or both. Receptorstherefore include full length intact native receptors containing anextracellular, transmembrane or cytoplasmic portion, as well astruncated forms or fragments thereof (e.g., an extracellular,transmembrane or cytoplasmic portion or subsequence of the receptoralone, or in combination). For example, a soluble receptor typicallylacks a transmembrane and may optionally also lack all or a part of thenative extracellular or cytoplasmic region (if present in nativereceptor). Such truncated receptor forms and fragments can retain atleast partial binding to a ligand.

Targeting and binding moiety domains of fusion constructs include orconsist of any entity that binds to a receptor, denoted a receptorligand, specifically or non-specifically. Non-limiting examples oftargeting and binding moieties therefore include hormone, a hormoneanalogue, a fragment of a hormone or hormone analogue that binds to ahormone receptor, a growth factor, growth factor analog, a fragment of agrowth factor or growth factor analogue that binds to a receptor, ahormone receptor or a ligand that binds to a hormone or to a hormonereceptor, and targeting and binding moieties that bind to a hormone, ahormone analogue, a fragment of a hormone or hormone analogue that bindsto a hormone receptor, a hormone receptor or a ligand that binds to ahormone or to a hormone receptor, growth factor, growth factor analogue,a fragment of a growth factor or growth factor analogue that binds to areceptor, a growth factor receptor or a ligand that binds to a growthfactor or to a growth factor receptor, etc.

Exemplary hormones useful as binding moieties includegonadotropin-releasing hormone I, gonadotropin-releasing hormone II,lamprey III luteinizing hormone releasing hormone, luteinizing hormonebeta chain, luteinizing hormone (LH), chorionic gonadotropin (CG),chorionic gonadotropin beta subunit (β- or beta-CG), melanocytestimulating hormone, estradiol, diethylstilbesterol, dopamine,somatostatin, follicle-stimulating hormone (FSH), glucocorticoids,estrogens, testosterone, androstenedione, dihydrotestosterone,dehydroepiandrosterone, progesterones, androgens and derivativesthereof. Exemplary hormone receptors useful as binding moieties includegonadotropin-releasing hormone I receptor, gonadotropin-releasinghormone II receptor, lamprey III luteinizing hormone releasing hormonereceptor, luteinizing hormone receptor, chorionic gonadotropin receptor,melanocyte stimulating hormone receptor, estradiol receptor, dopaminereceptor, somatostatin receptor, follicle-stimulating hormone (FSH)receptor, epidermal growth factor (EGF) receptor, growth hormone (GH)receptor, Her2-neu receptor, glucocorticoid hormone receptor, estrogenreceptor, testosterone receptor, progesterone receptor and androgenreceptor.

Exemplary growth factors include epidermal growth factor (EGF), growthhormone (GH), and Her2-neu. Exemplary growth factor receptors includeepidermal growth factor (EGF) receptor, growth hormone (GH) receptor,and Her2-neu receptor, IGF-1.

Specific non-limiting examples of targeting or binding moieties includeLHRH, LHRH functional (binding) fragments thereof, LHRH analogues, andβCG, βCG functional (binding) fragments thereof and βCG analogs. LHRH isa fully functional ligand and can elicit pharmacological effects throughligand receptor interaction, such as activation of signal transductionpathways. βCG-ala is a fragment of hCG that can bind to the cellmembrane without eliciting any pharmacological effect. Specificnon-limiting examples of targeting or binding moieties include orconsist of an amino acid sequence within or set forth as:SYAVALSAQAALARR (SEQ. ID NO. 8); SYAVALSAQAALARRA (SEQ. ID NO. 13),which are fragments of βCG, and QHWSYGLRPG (SEQ. ID NO. 9), which is anLHRH sequence.

Targeting and binding moities further include antigens for ligandsexpressed exclusively or preferentially in neoplastic, tumor or cancercells, and lymphatic or blood vessels associated with neoplastic, tumoror cancer cells. Such antigens can be conveniently referred to as “tumorassociated antigens,” or “TAA”, and include carcinoembryonic antigen(CEA), alpha-fetoprotein (AFP), prostate specific antigen (PSA),prostate specific membrane antigen (PSMA), CA-125 (residual epithelialovarian cancer), soluble Interleukin-2 (IL-2) receptor, RAGE-1,tyrosinase, MAGE-1, MAGE-2, NY-ESO-1, Melan-A/MART-1, glycoprotein (gp)75, gp100, beta-catenin, PRAME, MUM-1, ZFP161, Ubiquilin-1, HOX-B6,YB-1, Osteonectin, and ILF3, IGF-1, to name a few. Other antigens thatcan be targeted are CD19, CD20, CD23, CD27, CD28, CD30, CD33, CD40,CD52, CD56, CD70, CD154, immunoglobulin-like receptors etc).

Targeting and binding moities additionally include transferrin, folicacid and derivatives thereof (e.g., folate), and tumor necrosis factor(TNF) family members and TNF receptors, such as TNF-alpha, TNF-beta(lymphtoxin, LT), TRAIL, Fas, LIGHT, 41BB.

Fusion constructs in which a second domain includes or consists of atargeting or binding domain can bind to a cell that produces orexpresses an antigen, receptor or ligand, integrin, antibody or antigen,or TAA to which the second domain binds. Non-limiting examples of cellsinclude hyperproliferative cells and cells that exhibit aberrant orundesirable hyperproliferation. In particular non-limiting examples,such cells include non-metastatic and metastatic neoplastic, cancer,tumor and malignant cells, as well as disseminated neoplastic, cancer,tumor and malignant cells and dormant neoplastic, cancer, tumor andmalignant cells. Cells that express an antigen, receptor, ligand,integrin, TAA, etc., at elevated levels relative to normal ornon-hyperproliferating cells provide selectivity for such cells. Thus, atargeting or binding moiety can bind to an antigen, receptor, ligand,integrin or TAA that is expressed in or produced by a hyperproliferativecell (e.g., non-metastatic and metastatic neoplasias, cancers, tumorsand malignancies, and disseminated and dormant neoplastic, cancer, tumorand malignant cells), but not detectably expressed or is produced orexpressed at relatively lower levels by a normal ornon-hyperproliferative cell, thereby preferentially targetinghyperproliferative cells. Exemplary non-limiting cell and tissue typesthat express an antigen, receptor, ligand, integrin or TAA include abreast, ovarian, uterine, cervical, prostate, testicular, adrenal,pituitary, pancreatic, hepatic, gastrointestinal, skin, muscle orendometrial cell.

Additional examples of binding moieties include antibodies and antibodyfragments. An “antibody” refers to any monoclonal or polyclonalimmunoglobulin molecule, such as IgM, IgG, IgA, IgE, IgD, and anysubclass thereof. Exemplary subclasses for IgG are IgG₁, IgG₂, IgG₃ andIgG₄. Antibodies include those produced by or expressed on cells, suchas B cells. An antibody fragment or subsequence refers to a portion of afull length antibody that retains at least partial antigen bindingcapability of a comparison full length antibody. Exemplary antibodyfragments include Fab, Fab′, F(ab′)₂, Fv, Fd, single-chain Fv (scFv),disulfide-linked Fvs (sdFv), V_(L), V_(H), trispecific (Fab₃),bispecific (Fab₂), diabody ((V_(L)-V_(H))₂ or (V_(H)-V_(L))₂), triabody(trivalent), tetrabody (tetravalent), minibody ((scF_(V)-C_(H)3)₂),bispecific single-chain Fv (Bis-scFv), IgGdeltaCH2, scFv-Fc, (scFv)₂-Fc,or other antigen binding fragment of an intact immunoglobulin.

Fusion constructs include those with a first domain at theamino-terminus and a second domain at the carboxyl-terminus. Fusionconstructs also include those with a first domain at thecarboxyl-terminus and a second domain at the amino-terminus. Whereadditional domains are present (e.g., third, fourth, fifth, sixth,seventh, etc. domains), a first domain is positioned at the NH₂-terminusrelative to a second domain, or a second domain is positioned at theNH₂-terminus relative to a first domain.

Subsequences and amino acid substitutions of the various sequences setforth herein, such as, KFAKFAKKFAKFAKK (SEQ. ID NO. 1), KFAKFAKKFAKFAKKF(SEQ. ID NO. 2), KFAKFAKKFAKFAKKFA (SEQ. ID NO. 3), KFAKFAKKFAKFAKKFAK(SEQ. ID NO. 4), KFAKFAKKFAKFAKKFAKF (SEQ. ID NO. 5),KFAKFAKKFAKFAKKFAKFA (SEQ. ID NO. 6) and KFAKFAKKFAKFAKKFAKFAKKFAKFAK(SEQ. ID NO. 7), or a binding moiety, are also included. In particularembodiments, a subsequence of a first or second domain has at least 5 to10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35 or more amino acidresidues.

The invention therefore includes modifications or variations, such assubstitutions, additions or deletions of a first or second domain, orboth first and second domains. Thus, a fusion construct that includes apeptide sequence first or second domain can incorporate any number ofconservative or non-conservative amino acid substitutions, as long assuch substitutions do not destroy activity (lytic or binding) of firstor second domains. Thus, for example, a modified lytic portion (firstdomain) can retain at least partial lytic activity, such as cell killingor apoptosis, of an unmodified first domain, and a modified bindingmoiety or mimetic thereof can retain at least a partial binding activityof an unmodified binding moiety.

A “conservative substitution” is a replacement of one amino acid by abiologically, chemically or structurally similar residue. Biologicallysimilar means that the substitution is compatible with a biologicalactivity, e.g., lytic activity. Structurally similar means that theamino acids have side chains with similar length, such as alanine,glycine and serine, or having similar size, or the structure of a first,second or additional domain is maintained, such as an amphipathic alphhelix. Chemical similarity means that the residues have the same chargeor are both hydrophilic or hydrophobic. Particular examples include thesubstitution of one hydrophobic residue, such as isoleucine, valine,leucine or methionine for another, or the substitution of one polarresidue for another, such as the substitution of arginine for lysine,glutamic for aspartic acids, or glutamine for asparagine, serine forthreonine, etc. Routine assays can be used to determine whether a fusionconstruct variant has activity, e.g., lytic activity or bindingactivity.

Specific examples include a substitution or deletion of one or moreamino acid (e.g., 1-3, 3-5, 5-10, 10-20, or more) residues of a peptidefirst or second domain. A modified fusion construct can have a peptidesequence with 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, ormore identity to a reference sequence (e.g., a first domain, such asKFAKFAKKFAKFAKK (SEQ. ID NO. 1), KFAKFAKKFAKFAKKF (SEQ. ID NO. 2),KFAKFAKKFAKFAKKFA (SEQ. ID NO. 3), KFAKFAKKFAKFAKKFAK (SEQ. ID NO. 4),KFAKFAKKFAKFAKKFAKF (SEQ. ID NO. 5), KFAKFAKKFAKFAKKFAKFA (SEQ. ID NO.6) or KFAKFAKKFAKFAKKFAKFAKKFAKFAK (SEQ. ID NO. 7), or a second domainsuch as a binding moiety).

In a particular embodiment, a fusion construct includes a peptide firstdomain that includes or consists of a 12, 13, 15, 16, 17, 18, 19, 20,22, 23, 24, 25, 26, 27 or 28 residue L- or D-amino acid sequence thatincludes a peptide selected from KFAKFAKKFAKFAKK (SEQ. ID NO. 1),KFAKFAKKFAKFAKKF (SEQ. ID NO. 2), KFAKFAKKFAKFAKKFA (SEQ. ID NO. 3),KFAKFAKKFAKFAKKFAK (SEQ. ID NO. 4), KFAKFAKKFAKFAKKFAKF (SEQ. ID NO. 5),KFAKFAKKFAKFAKKFAKFA (SEQ. ID NO. 6) and KFAKFAKKFAKFAKKFAKFAKKFAKFAK(SEQ. ID NO. 7) having one or more of the K residues substituted with anF or L residue, one or more of the F residues substituted with a K, A orL residue, or one or more of the A residues substituted with a K, F or Lresidue. In another particular embodiment, a fusion construct includes apeptide first domain consisting of an L- or D-amino acid sequenceselected from KFAKFAKKFAKFAKK (SEQ. ID NO. 1), KFAKFAKKFAKFAKKF (SEQ. IDNO. 2), KFAKFAKKFAKFAKKFA (SEQ. ID NO. 3), KFAKFAKKFAKFAKKFAK (SEQ. IDNO. 4), KFAKFAKKFAKFAKKFAKF (SEQ. ID NO. 5), KFAKFAKKFAKFAKKFAKFA (SEQ.ID NO. 6) and KFAKFAKKFAKFAKKFAKFAKKFAKFAK (SEQ. ID NO. 7) having one ormore of the K residues substituted with an F or L residue, one or moreof the F residues substituted with a K, A or L residue, or one or moreof the A residues substituted with a K, F or L residue; and a peptidesecond domain that includes or consists of a binding moiety. In furtherparticular embodiment, a fusion construct includes or consists of apeptide first domain consisting of an L- or D-amino acid sequenceselected from KFAKFAKKFAKFAKK (SEQ. ID NO. 1), KFAKFAKKFAKFAKKF (SEQ. IDNO. 2), KFAKFAKKFAKFAKKFA (SEQ. ID NO. 3), KFAKFAKKFAKFAKKFAK (SEQ. IDNO. 4), KFAKFAKKFAKFAKKFAKF (SEQ. ID NO. 5), KFAKFAKKFAKFAKKFAKFA (SEQ.ID NO. 6) and KFAKFAKKFAKFAKKFAKFAKKFAKFAK (SEQ. ID NO. 7) having one ormore of the K residues substituted with any of an F or L residue, one ormore of the F residues substituted with any of a K, A or L residue, orone or more of the A residues substituted with any of a K, F or Lresidue, and a peptide second domain consisting of a 1-25 L- or D-aminoacid sequence (e.g., binding moiety) distinct from the first domain.

The term “identity” and “homology” and grammatical variations thereofmean that two or more referenced entities are the same. Thus, where twoamino acid sequences are identical, they have the same amino acidsequence. “Areas, regions or domains of identity” mean that a portion oftwo or more referenced entities are the same. Thus, where two amino acidsequences are identical or homologous over one or more sequence regions,they share identity in these regions. The term “complementary,” whenused in reference to a nucleic acid sequence means the referencedregions are 100% complementary, i.e., exhibit 100% base pairing with nomismatches.

Due to variation in the amount of sequence conservation betweenstructurally and functionally related proteins, the amount of sequenceidentity required to retain a function or activity (e.g., lytic orbinding) depends upon the protein, the region and the function oractivity of that region. For example, for a lytic peptide sequencemultiple PNNPNNP (SEQ. ID NO. 14) sequence repeat patterns or motifs canbe present, but one or more interrupted or non-interrupted PNNPNNP (SEQ.ID NO. 14) sequence repeat patterns or motifs need not be present.

The extent of identity between two sequences can be ascertained using acomputer program and mathematical algorithm known in the art. Suchalgorithms that calculate percent sequence identity (homology) generallyaccount for sequence gaps and mismatches over the comparison region. Forexample, a BLAST (e.g., BLAST 2.0) search algorithm (see, e.g., Altschulet al., J. Mol. Biol. 215:403 (1990), publicly available through NCBI)has exemplary search parameters as follows: Mismatch −2; gap open 5; gapextension 2. For polypeptide sequence comparisons, a BLASTP algorithm istypically used in combination with a scoring matrix, such as PAM100, PAM250, BLOSUM 62 or BLOSUM 50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCHsequence comparison programs are also used to quantitate the extent ofidentity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988);Pearson, Methods Mol Biol. 132:185 (2000); and Smith et al., J. Mol.Biol. 147:195 (1981)). Programs for quantitating protein structuralsimilarity using Delaunay-based topological mapping have also beendeveloped (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

Individual residues and first, second and additional domains can bejoined by a covalent or a non-covalent bond. Non-limiting examples ofcovalent bonds are amide bonds, non-natural and non-amide chemicalbonds, which include, for example, glutaraldehyde, N-hydroxysuccinimideesters, bifunctional maleimides, N,N′-dicyclohexylcarbodiimide (DCC) orN,N′-diisopropylcarbodiimide (DIC). Linking groups alternative to amidebonds include, for example, ketomethylene (e.g., —C(═O)—CH₂— for—C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin (CH═CH), ether(CH₂—O), thioether (CH₂—S), tetrazole (CN₄—), thiazole, retroamide,thioamide, or ester (see, e.g., Spatola (1983) in Chemistry andBiochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357,“Peptide and Backbone Modifications,” Marcel Decker, NY).

First and second domains can be fused or joined immediately adjacent toeach other by a covalent or a non-covalent bond. First and seconddomains can be separated by an intervening region, such as a hinge,spacer or linker positioned between a first and a second domain. In oneembodiment, a first and second domain are joined by a carbon chain.Multi-carbon chains include carboxylic acids (e.g., dicarboxylic acids)such as glutaric acid, succinic acid and adipic acid.

In another embodiment, a first and second domain are joined by an aminoacid, peptide or a non-peptide hinge, spacer or linker positionedbetween the first and second domains. Peptide hinge, spacer or linkersequences can be any length, but typically range from about 1-10, 10-20,20-30, 30-40, or 40-50 amino acid residues. In particular embodiments, apeptide hinge, spacer or linker positioned between a first and seconddomain is from 1 to 25 L- or D-amino acid residues, or 1 to 6 L- orD-amino acid residues. Particular amino acid residues that are includedin sequences positioned between the first and second domains include oneor more of or C, A, S or G amino acid residues. Specific non-limitingexamples of peptides positioned between the first and second domainsinclude a sequence within or set forth as: GSGGS (SEQ. ID NO. 10), ASAAS(SEQ. ID NO. 11), or CCCCCC (SEQ. ID NO. 12). Derivatives of amino acidsand peptides can be positioned between the first and second domain. Aspecific non-limiting example of an amino acid derivative is a lysinederivative, or a 6 carbon linker such as α-amino-caproic acid.

Fusion constructs with or without a hinge, spacer or linker, or a third,fourth, fifth, sixth, seventh, etc. domain can be entirely composed ofnatural amino acids or synthetic, non-natural amino acids or amino acidanalogues, or can include derivatized forms. In various embodiments, afusion construct includes in a first or second domain one or moreD-amino acids substituted for L-amino acids, mixtures of D-amino acidsand L-amino acids, or a sequence composed entirely of D-amino acidresidues.

Fusion constructs can contain any combination of non-natural structuralcomponents, which are typically from three structural groups: a) residuelinkage groups other than the natural amide bond (“peptide bond”)linkages; b) non-natural residues in place of naturally occurring aminoacid residues; or c) residues which induce secondary structural mimicry,i.e., induce or stabilize a secondary structure, e.g., an alpha helixconformation. Fusion constructs include cyclic structures such as anend-to-end amide bond between the amino and carboxy-terminus of themolecule or intra- or inter-molecular disulfide bond(s). Fusionconstructs may be modified in vitro or in vivo, e.g.,post-translationally modified to include, for example, sugar orcarbohydrate residues, phosphate groups, fatty acids, lipids, etc.

Specific examples of an addition include a third, fourth, fifth, sixthor seventh domain. Fusion constructs with a first and second domaintherefore include one or more additional domains (third, fourth, fifth,sixth, seventh, etc.) covalently linked thereto to impart a distinct orcomplementary function or activity. Exemplary additional domains includedomains facilitating isolation, which include, for example, metalchelating peptides such as polyhistidine tracts and histidine-tryptophanmodules that allow purification on immobilized metals; protein A domainsthat allow purification on immobilized immunoglobulin; and domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle Wash.). Optional inclusion of a cleavable sequence such asFactor Xa or enterokinase between a purification domain and the fusionconstruct can be used to facilitate purification. For example, anexpression vector can include a fusion construct-encoding nucleic acidsequence linked to six histidine residues followed by a thioredoxin andan enterokinase cleavage site. The histidine residues facilitatedetection and purification of the fusion construct while theenterokinase cleavage site provides a means for purifying the constructfrom the remainder of the protein (see e.g., Kroll, DNA Cell. Biol.12:441 (1993)).

Fusion construct activity can be affected by various factors andtherefore fusion constructs can be designed or optimized by taking intoconsideration one or more of these factors. Such factors include, forexample, length of a fusion construct, which can affect toxicity tocells. In particular, increased cytotoxicity was observed whenPhor21-βCG-ala and Phor21 were compared to Phor14-βCG-ala. Cell killingactivity of alpha helix forming lytic peptide domains can also depend onthe stability of the helix. Hinge and spacers can affect membraneinteraction of a first domain and the helical structure of a peptidelytic domain. For example, shorter fusion constructs, such as constructsless than 21 amino acids that optionally include a spacer or hinge, canexhibit increased cytotoxicity due to increased helix stability. Inparticular, spacers such as ASAAS (SEQ. ID NO. 11) and 6 aminocaproicacid tend to increase toxicity of shorter fusion constructs. The chargeof lytic peptide domains, which is determined in part by the particularamino acid residues present in the domain, also affects cell killingpotency.

The positioning of the binding moiety relative to the lytic domain (N-or C-terminus) also can affect cell killing activity of fusionconstructs. For example, a binding moiety positioned at the C-terminusrelative to the lytic domain had greater cell killing activity than ifpositioned at the N-terminus relative to the lytic domain.

Fusion construct in vivo half-life can be increased by constructingfusion construct peptide domains with one or more non-naturallyoccurring amino acids or derivatives. For example, fusion constructswith D-amino acids (e.g., up to 30% or more of all residues areD-enantiomers) are resistant to serum proteolysis and therefore can beactive for longer times thereby increasing in vivo potency. Furthermore,constructing fusion construct peptide domains with one or morenon-naturally occurring amino acids or derivatives can reduce hemolyticactivity. Such fusion constructs with D-enantiomers also have a greatertendency to be monomeric in solution-they do not significantlyaggregate.

In accordance with the invention, there are provided fusion constructsthat have greater anti-cell proliferative activity than one or more ofPhor21-βCG-ala, Phor21-GSGGS-βCG-ala, Phor21-ASAAS-βCG-ala, or Phor14-βCG-ala, as ascertained by a lower IC₅₀ value, which represents theamount of fusion construct required to achieve cell cytotoxicity. Inaccordance with the invention, there are also provided fusion constructsthat have less hemolytic activity, as represented by IC₅₀/HA₅₀(hemolytic activity) ratio, than Phor21-βCG-ala, Phor21-GSGGS-βCG-ala,Phor21-ASAAS-βCG-ala, or Phor 14-βCG-ala. In accordance with theinvention, there are further provided fusion constructs that have ahemolytic activity, as represented by IC₅₀/HA₅₀ (hemolytic activity)ratio, of less than about 0.02, 0.01, or 0.005. Representative assayconditions for determining cell cytotoxicty and hemolytic activity areset forth in Example 1.

Peptides and peptidomimetics can be produced and isolated using methodsknown in the art. Peptides can be synthesized, whole or in part, usingchemical methods known in the art (see, e.g., Caruthers (1980). NucleicAcids Res. Symp. Ser. 215; Horn (1980); and Banga, A. K., TherapeuticPeptides and Proteins, Formulation, Processing and Delivery Systems(1995) Technomic Publishing Co., Lancaster, Pa.). Peptide synthesis canbe performed using various solid-phase techniques (see, e.g., RobergeScience 269:202 (1995); Merrifield, Methods Enzymol. 289:3(1997)) andautomated synthesis may be achieved, e.g., using the ABI 431A PeptideSynthesizer (Perkin Elmer) in accordance with the manufacturer'sinstructions. Peptides and peptide mimetics can also be synthesizedusing combinatorial methodologies. Synthetic residues and polypeptidesincorporating mimetics can be synthesized using a variety of proceduresand methodologies known in the art (see, e.g., Organic SynthesesCollective Volumes, Gilman, et al. (Eds) John Wiley & Sons, Inc., NY).Modified peptides can be produced by chemical modification methods (see,for example, Belousov, Nucleic Acids Res. 25:3440 (1997); Frenkel, FreeRadic. Biol. Med. 19:373 (1995); and Blommers, Biochemistry 33:7886(1994).

The invention further provides nucleic acids encoding the fusionconstructs of the invention and vectors that include nucleic acid thatencodes fusion constructs. In a particular embodiment, a nucleic acidencodes a fusion construct that includes a first domain consisting of a12, 13, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27 or 28 residueamino acid sequence that includes a peptide sequence selected fromKFAKFAKKFAKFAKK (SEQ. ID NO. 1), KFAKFAKKFAKFAKKF (SEQ. ID NO. 2),KFAKFAKKFAKFAKKFA (SEQ. ID NO. 3), KFAKFAKKFAKFAKKFAK (SEQ. ID NO. 4),KFAKFAKKFAKFAKKFAKF (SEQ. ID NO. 5), KFAKFAKKFAKFAKKFAKFA (SEQ. ID NO.6) and KFAKFAKKFAKFAKKFAKFAKKFAKFAK (SEQ. ID NO. 7), and a second domainthat includes or consists of a targeting or binding moiety. In anotherembodiment, a nucleic acid encodes a fusion construct that includes afirst domain consisting of an amino acid sequence selected fromKFAKFAKKFAKFAKK (SEQ. ID NO. 1), KFAKFAKKFAKFAKKF (SEQ. ID NO. 2),KFAKFAKKFAKFAKKFA (SEQ. ID NO. 3), KFAKFAKKFAKFAKKFAK (SEQ. ID NO. 4),KFAKFAKKFAKFAKKFAKF (SEQ. ID NO. 5), KFAKFAKKFAKFAKKFAKFA (SEQ. ID NO.6) and KFAKFAKKFAKFAKKFAKFAKKFAKFAK (SEQ. ID NO. 7), and a second domainthat includes or consist of a targeting or binding moiety. In a furtherembodiment, a nucleic acid encodes a fusion construct that includes orconsists of a first domain consisting of an amino acid sequence selectedfrom KFAKFAKKFAKFAKK (SEQ. ID NO. 1), KFAKFAKKFAKFAKKF (SEQ. ID NO. 2),KFAKFAKKFAKFAKKFA (SEQ. ID NO. 3), KFAKFAKKFAKFAKKFAK (SEQ. ID NO. 4),KFAKFAKKFAKFAKKFAKF (SEQ. ID NO. 5), KFAKFAKKFAKFAKKFAKFA (SEQ. ID NO.6) and KFAKFAKKFAKFAKKFAKFAKKFAKFAK (SEQ. ID NO. 7), and a second domainconsisting of a 1-25 amino acid sequence (e.g., targeting or bindingmoiety) distinct from said first domain.

Nucleic acid, which can also be referred to herein as a gene,polynucleotide, nucleotide sequence, primer, oligonucleotide or proberefers to natural or modified purine- and pyrimidine-containing polymersof any length, either polyribonucleotides or polydeoxyribonucleotides ormixed polyribo-polydeoxyribo nucleotides and α-anomeric forms thereof.The two or more purine- and pyrimidine-containing polymers are typicallylinked by a phosphoester bond or analog thereof. The terms can be usedinterchangeably to refer to all forms of nucleic acid, includingdeoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The nucleicacids can be single strand, double, or triplex, linear or circular.Nucleic acids include genomic DNA, cDNA, and antisense. RNA nucleic acidcan be spliced or unspliced mRNA, rRNA, tRNA or antisense. Nucleic acidsinclude naturally occurring, synthetic, as well as nucleotide analoguesand derivatives.

As a result of the degeneracy of the genetic code, nucleic acids includesequences degenerate with respect to sequences encoding fusionconstructs of the invention. Thus, degenerate nucleic acid sequencesencoding fusion constructs are provided.

Nucleic acid can be produced using any of a variety of known standardcloning and chemical synthesis methods, and can be altered intentionallyby site-directed mutagenesis or other recombinant techniques known toone skilled in the art. Purity of polynucleotides can be determinedthrough sequencing, gel electrophoresis, UV spectrometry.

Nucleic acids may be inserted into a nucleic acid construct in whichexpression of the nucleic acid is influenced or regulated by an“expression control element,” referred to herein as an “expressioncassette.” The term “expression control element” refers to one or morenucleic acid sequence elements that regulate or influence expression ofa nucleic acid sequence to which it is operatively linked. An expressioncontrol element can include, as appropriate, promoters, enhancers,transcription terminators, gene silencers, a start codon (e.g., ATG) infront of a protein-encoding gene, etc.

An expression control element operatively linked to a nucleic acidsequence controls transcription and, as appropriate, translation of thenucleic acid sequence. The term “operatively linked” refers to ajuxtaposition wherein the referenced components are in a relationshippermitting them to function in their intended manner. Typicallyexpression control elements are juxtaposed at the 5′ or the 3′ ends ofthe genes but can also be intronic.

Expression control elements include elements that activate transcriptionconstitutively, that are inducible (i.e., require an external signal foractivation), or derepressible (i.e., require a signal to turntranscription off; when the signal is no longer present, transcriptionis activated or “derepressed”). Also included in the expressioncassettes of the invention are control elements sufficient to rendergene expression controllable for specific cell-types or tissues (i.e.,tissue-specific control elements). Typically, such elements are locatedupstream or downstream (i.e., 5′ and 3′) of the coding sequence.Promoters are generally positioned 5′ of the coding sequence. Promoters,produced by recombinant DNA or synthetic techniques, can be used toprovide for transcription of the polynucleotides of the invention. A“promoter” is meant a minimal sequence element sufficient to directtranscription.

Nucleic acids may be inserted into a plasmid for propagation into a hostcell and for subsequent genetic manipulation if desired. A plasmid is anucleic acid that can be stably propagated in a host cell; plasmids mayoptionally contain expression control elements in order to driveexpression of the nucleic acid. A vector is used herein synonymouslywith a plasmid and may also include an expression control element forexpression in a host cell. Plasmids and vectors generally contain atleast an origin of replication for propagation in a cell and a promoter.Plasmids and vectors are therefore useful for genetic manipulation offusion construct encoding nucleic acids, producing fusion constructs orantisense nucleic acid, and expressing fusion constructs in host cellsand organisms, for example.

Bacterial system promoters include T7 and inducible promoters such as pLof bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) andtetracycline responsive promoters. Insect cell system promoters includeconstitutive or inducible promoters (e.g., ecdysone). Mammalian cellconstitutive promoters include SV40, RSV, bovine papilloma virus (BPV)and other virus promoters, or inducible promoters derived from thegenome of mammalian cells (e.g., metallothionein IIA promoter; heatshock promoter) or from mammalian viruses (e.g., the adenovirus latepromoter; the inducible mouse mammary tumor virus long terminal repeat).Alternatively, a retroviral genome can be genetically modified forintroducing and directing expression of a fusion construct inappropriate host cells.

Expression systems further include vectors designed for in vivo use.Particular non-limiting examples include adenoviral vectors (U.S. Pat.Nos. 5,700,470 and 5,731,172), adeno-associated vectors (U.S. Pat. No.5,604,090), herpes simplex virus vectors (U.S. Pat. No. 5,501,979),retroviral vectors (U.S. Pat. Nos. 5,624,820, 5,693,508 and 5,674,703),BPV vectors (U.S. Pat. No. 5,719,054) and CMV vectors (U.S. Pat. No.5,561,063).

Yeast vectors include constitutive and inducible promoters (see, e.g.,Ausubel et al., In: Current Protocols in Molecular Biology, Vol. 2, Ch.13, ed., Greene Publish. Assoc. & Wiley Interscience, 1988; Grant et al.Methods in Enzymology, 153:516 (1987), eds. Wu & Grossman; BitterMethods in Enzymology, 152:673 (1987), eds. Berger & Kimmel, Acad.Press, N.Y.; and, Strathern et al., The Molecular Biology of the YeastSaccharomyces (1982) eds. Cold Spring Harbor Press, Vols. I and II). Aconstitutive yeast promoter such as ADH or LEU2 or an inducible promotersuch as GAL may be used (R. Rothstein In: DNA Cloning, A PracticalApproach, Vol. 11, Ch. 3, ed. D. M. Glover, IRL Press, Wash., D.C.,1986). Vectors that facilitate integration of foreign nucleic acidsequences into a yeast chromosome, via homologous recombination forexample, are known in the art. Yeast artificial chromosomes (YAC) aretypically used when the inserted polynucleotides are too large for moreconventional vectors (e.g., greater than about 12 Kb).

Expression vectors also can contain a selectable marker conferringresistance to a selective pressure or identifiable marker (e.g.,beta-galactosidase), thereby allowing cells having the vector to beselected for, grown and expanded. Alternatively, a selectable marker canbe on a second vector that is cotransfected into a host cell with afirst vector containing a nucleic acid encoding a fusion construct.

Selection systems include but are not limited to herpes simplex virusthymidine kinase gene (Wigler et al., Cell 11:223 (1977)),hypoxanthine-guanine phosphoribosyltransferase gene (Szybalska et al.,Proc. Natl. Acad. Sci. USA 48:2026 (1962)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes whichcan be employed in tk-, hgprt- or aprt-cells, respectively.Additionally, antimetabolite resistance can be used as the basis ofselection for dhfr, which confers resistance to methotrexate (O'Hare etal., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); the gpt gene, whichconfers resistance to mycophenolic acid (Mulligan et al., Proc. Natl.Acad. Sci. USA 78:2072 (1981)); neomycin gene, which confers resistanceto aminoglycoside G-418 (Colberre-Garapin et al., J. Mol. Biol.150:1(1981)); puromycin; and hygromycin gene, which confers resistanceto hygromycin (Santerre et al., Gene 30:147 (1984)). Additionalselectable genes include trpB, which allows cells to utilize indole inplace of tryptophan; hisD, which allows cells to utilize histinol inplace of histidine (Hartman et al., Proc. Natl. Acad. Sci. USA 85:8047(1988)); and ODC (ornithine decarboxylase), which confers resistance tothe ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine,DEMO (McConlogue (1987) In: Current Communications in Molecular Biology,Cold Spring Harbor Laboratory).

Host cells that express fusion constructs, and host cells transformedwith nucleic acids encoding fusion constructs and vectors including anucleic acid that encodes the fusion construct are also provided. In oneembodiment, a host cell is a prokaryotic cell. In another embodiment, ahost cell is a eukaryotic cell. In various aspects, the eukaryotic cellis a yeast or mammalian (e.g., human, primate, etc.) cell.

As used herein, a “host cell” is a cell into which a nucleic acid isintroduced that can be propagated, transcribed, or encoded fusionconstruct expressed. The term also includes any progeny or subclones ofthe host cell. Host cells include cells that express fusion constructand cells that do not express fusion construct. Host cells that do notexpress a fusion construct are used to propagate nucleic acid or vectorwhich includes a nucleic acid encoding a fusion construct or anantisense.

Host cells include but are not limited to microorganisms such asbacteria and yeast; and plant, insect and mammalian cells. For example,bacteria transformed with recombinant bacteriophage nucleic acid,plasmid nucleic acid or cosmid nucleic acid expression vectors; yeasttransformed with recombinant yeast expression vectors; plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid); insect cell systems infected with recombinant virus expressionvectors (e.g., baculovirus); and animal cell systems infected withrecombinant virus expression vectors (e.g., retroviruses, adenovirus,vaccinia virus), or transformed animal cell systems engineered fortransient or stable propagation or expression.

Fusion constructs, nucleic acids encoding fusion constructs, vectors andhost cells expressing fusion constructs or transformed with nucleicacids encoding fusion constructs and antisense include isolated andpurified forms. The term “isolated,” when used as a modifier of aninvention composition, means that the composition is made by the hand ofman or is separated, substantially completely or at least in part, fromthe naturally occurring in vivo environment. Generally, an isolatedcomposition is substantially free of one or more materials with which itnormally associates with in nature, for example, one or more protein,nucleic acid, lipid, carbohydrate, cell membrane. The term “isolated”does not exclude alternative physical forms of the composition, such asmultimers/oligomers, variants, modifications or derivatized forms, orforms expressed in host cells produced by the hand of man. The term“isolated” also does not exclude forms (e.g., pharmaceuticalformulations and combination compositions) in which there arecombinations therein, any one of which is produced by the hand of man.

An “isolated” composition can also be “purified” when free of some, asubstantial number of, most or all of the materials with which ittypically associates with in nature. Thus, an isolated fusion constructthat also is substantially pure does not include polypeptides orpolynucleotides present among millions of other sequences, such asproteins of a protein library or nucleic acids in a genomic or cDNAlibrary, for example. A “purified” composition can be combined with oneor more other molecules.

In accordance with the invention, there are provided mixtures of fusionconstructs and combination compositions. In one embodiment, a mixtureincludes one or more fusion constructs and a pharmaceutically acceptablecarrier or excipient. In another embodiment, a mixture includes one ormore fusion constructs and an anti-cell proliferative, anti-tumor,anti-cancer, or anti-neoplastic treatment or agent. In a furtherembodiment, a mixture includes one or more fusion constructs and animmune enhancing agent. Combinations, such as one or more fusionconstructs in a pharmaceutically acceptable carrier or excipient, withone or more of an anti-cell proliferative, anti-tumor, anti-cancer, oranti-neoplastic treatment or agent, and an immune enhancing treatment oragent, are also provided.

Fusion constructs of the invention, such as polypeptides having an aminoacid sequence including a first lytic domain and a second binding moietydomain, can be used to target cells for lysis, cell death or apoptosis.Such cells can be selectively targeted. For example a cell thatexpresses a receptor, ligand, antigen or antibody can be targeted by afusion construct and thereby be preferentially killed compared to cellsthat express less of the receptor, ligand, antigen or antibody.

In accordance with the invention, there are provided methods of reducingor inhibiting proliferation of a cell, and methods of reducing orinhibiting cell proliferation. In one embodiment, a method includescontacting a cell with a fusion construct in an amount sufficient toreduce or inhibit proliferation of the cell. In another embodiment, amethod includes contacting a cell with a fusion construct in an amountsufficient to reduce or inhibit cell proliferation.

Also provided are methods of reducing or inhibiting proliferation of ahyperproliferative cell, and methods of reducing or inhibitingproliferation of hyperproliferating cells. In one embodiment, a methodincludes contacting a hyperproliferative cell or hyperproliferatingcells with a fusion construct in an amount sufficient to reduce orinhibit proliferation.

Further provided are methods of reducing or inhibiting proliferation ofa non-metastatic or metastatic neoplastic, cancer, tumor and malignantcell. In one embodiment, a method includes contacting a neoplastic,cancer, tumor or malignant cell with a fusion construct in an amountsufficient to reduce or inhibit proliferation of the cell.

Still further provided are methods of reducing or inhibitingproliferation of a dormant or non-dividing non-metastatic or metastaticneoplastic, cancer, tumor and malignant cell. In one embodiment, amethod includes contacting a dormant or non-dividing neoplastic, cancer,tumor or malignant cell with a fusion construct in an amount sufficientto reduce or inhibit proliferation of the dormant or non-dividing cell.

Additionally provided are methods of selectively reducing or inhibitingproliferation of a cell (e.g., a hyperproliferating cell) that expressesa receptor, ligand, antibody or antigen. In one embodiment, a methodincludes contacting the cell with a fusion construct in an amountsufficient to reduce or inhibit proliferation of the cell (e.g.,hyperproliferating cell), wherein the binding moiety of said peptidebinds to the receptor, ligand, antibody or antigen expressed by thecell.

Yet additionally provided are methods of selectively reducing orinhibiting proliferation of a neoplastic, tumor, cancer or malignantcell that expresses a receptor, ligand, antibody or antigen. In oneembodiment, a method includes contacting the cell with a fusionconstruct in an amount sufficient to reduce or inhibit proliferation ofthe neoplastic, tumor, cancer or malignant cell, wherein the bindingmoiety of said fusion construct binds to the receptor, ligand, antibodyor antigen expressed by the cell.

The term “contacting” means direct or indirect binding or interactionbetween two or more entities (e.g., between a fusion construct and acell). Contacting as used herein includes in solution, in solid phase,in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can bereferred to as administering, or administration.

Cells to target for reducing or inhibiting proliferation,non-selectively or selectively, include cells that express any moleculeto which the binding moiety of the fusion construct binds. Exemplarycells include a cell that expresses a receptor (e.g., a hormonereceptor, growth factor receptor, a cytokine receptor, a chemokinereceptor), ligand (e.g., a hormone, growth factor, cytokine, chemokine)or antibody or an antigen, or an integrin or integrin receptor (peptidescontaining “RGD” sequence motif), or a component present inextracellular matrix (ECM), such as mono-, di- or oligo-saccharides,sialic acid, galactose, mannose, fucose, acetylneuraminic acid, peptidescontaining “RGD” sequence motif, etc.

Target cells include cells that express a sex or gonadal steroid hormoneor a sex or gonadal steroid hormone receptor. Target cells also includecells that express a receptor that binds to gonadotropin-releasinghormone I, gonadotropin-releasing hormone II, lamprey III luteinizinghormone releasing hormone, luteinizing hormone, chorionic gonadotropin,melanocyte stimulating hormone, estradiol, diethylstilbesterol,dopamine, somatostatin, follicle-stimulating hormone (FSH),glucocorticoid, estrogen, testosterone, androstenedione,dihydrotestosterone, dehydroepiandrosterone, progesterone, androgen,epidermal growth factor (EGF), Her2/neu, vitamin H, folic acid or aderivative thereof (e.g., folate), transferrin, thyroid stimulatinghormone (TSH), endothelin, bombesin, growth hormone, vasoactiveintestinal peptide, lactoferrin, an integrin (e.g., alpha-5 beta 3 oralpha-5 beta 1 integrin), nerve growth factor, CD19, CD20, CD23, CD27,CD28, CD30, CD33, CD40, CD52, CD56, CD70, CD154, immunoglobulin-likereceptors, ROR1, IGF-1, carcinoembryonic antigen (CEA), prostatespecific antigen (PSA), prostate specific membrane antigen (PSMA),transforming growth factor alpha, transforming growth factor beta,insulin-like growth factor, vascular endothelial growth factor, insulin,ceruloplasmin, or HIV-tat.

Target cells further include cells that express a receptor that binds toa sex or gonadal steroid hormone or a sex or gonadal steroid hormonereceptor. Target cells moreover include cells that express a receptorthat binds to gonadotropin-releasing hormone I, gonadotropin-releasinghormone II, lamprey III luteinizing hormone releasing hormone,luteinizing hormone beta chain, luteinizing hormone, chorionicgonadotropin, chorionic gonadotropin beta subunit, melanocytestimulating hormone, estradiol, diethylstilbesterol, dopamine,somatostatin, follicle-stimulating hormone (FSH), glucocorticoid,glucocorticoid, estrogen, testosterone, androstenedione,dihydrotestosterone, dehydroepiandrosterone, progesterone, androgen,epidermal growth factor (EGF), Her2/neu, vitamin H, folic acid or aderivative thereof (e.g., folate), transferrin, thyroid stimulatinghormone (TSH), endothelin, bombesin, growth hormone, vasoactiveintestinal peptide, lactoferrin, an integrin (e.g., alpha-5 beta 3 oralpha-5 beta 1 integrin), nerve growth factor, CD19, CD20, CD23, CD27,CD28, CD30, CD33, CD40, CD52, CD56, CD70, CD154, immunoglobulin-likereceptors, ROR1, IGF-1, carcinoembryonic antigen (CEA), prostatespecific antigen (PSA), prostate specific membrane antigen (PSMA),transforming growth factor alpha, transforming growth factor beta,insulin, ceruloplasmin, HIV-tat, or an analogue thereof (e.g.,mifepristone, flutaminde, lupron, zxoladex, supprelin, synateltriptorelin, buserelin, centrorelix, ganirelix, abarelix, antide,teverelix or degarelix (Fe200486)).

Cells to target for reducing or inhibiting proliferation,non-selectively or selectively, additionally include cells that express“tumor associated antigens,” such as carcinoembryonic antigen (CEA),alpha-fetoprotein (AFP), prostate specific antigen (PSA), prostatespecific membrane antigen (PSMA), CA 125 (residual epithelial ovariancancer), soluble Interleukin-2 (IL-2) receptor, RAGE-1, tyrosinase,MAGE-1, MAGE-2, NY-ESO-1, Melan-A/MART-1, glycoprotein (gp) 75, gp100,beta-catenin, PRAME, MUM-1, ZFP161, Ubiquilin-1, HOX-B6, YB-1,Osteonectin, and ILF3. Cells to target for reducing or inhibitingproliferation, non-selectively or selectively, yet additionally includecells that express transferrin, folic acid and derivatives thereof(e.g., folate), and a tumor necrosis factor (TNF) family member or, suchas TNF-alpha, TNF-beta (lymphtoxin, LT), TRAIL, Fas, LIGHT, and 41BB,and receptors therefore.

Fusion constructs and methods of the invention are also applicable totreating undesirable or aberrant cell proliferation andhyperproliferative disorders. Thus, in accordance with the invention,methods of treating undesirable or aberrant cell proliferation andhyperproliferative disorders are provided. In one embodiment, a methodincludes administering to a subject (in need of treatment) an amount ofa fusion construct sufficient to treat the undesirable or aberrant cellproliferation or the hyperproliferative disorder.

The term “hyperproliferative disorder” refers to any undesirable oraberrant cell survival (e.g., failure to undergo programmed cell deathor apoptosis), growth or proliferation. Such disorders include benignhyperplasias, non-metastatic and metastatic neoplasias, cancers, tumorsand malignancies. Undesirable or aberrant cell proliferation andhyperproliferative disorders can affect any cell, tissue, organ in asubject. Undesirable or aberrant cell proliferation andhyperproliferative disorders can be present in a subject, locally,regionally or systemically. A hyperproliferative disorder can arise froma multitude of tissues and organs, including but not limited to breast,lung (e.g., small cell or non-small cell), thyroid, head and neck,brain, nasopharynx, throat, nose or sinuses, lymphoid, adrenal gland,pituitary gland, thyroid, lymph, gastrointestinal (mouth, esophagus,stomach, duodenum, ileum, jejunum (small intestine), colon, rectum),genito-urinary tract (uterus, ovary, vagina cervix, endometrium,fallopian tube, bladder, testicle, penis, prostate), kidney, pancreas,liver, bone, bone marrow, lymph, blood, muscle, skin, and stem cells,which may or may not metastasize to other secondary sites, regions orlocations.

Fusion constructs and methods of the invention are also applicable tometastatic or non-metastatic tumor, cancer, malignancy or neoplasia ofany cell, organ or tissue origin. Such disorders can affect virtuallyany cell or tissue type, e.g., carcinoma, sarcoma, melanoma, neural, andreticuloendothelial or haematopoietic neoplastic disorders (e.g.,myeloma, lymphoma or leukemia).

As used herein, the terms “neoplasia” and “tumor” refer to a cell orpopulation of cells whose growth, proliferation or survival is greaterthan growth, proliferation or survival of a normal counterpart cell,e.g. a cell proliferative or differentiative disorder. A tumor is aneoplasia that has formed a distinct mass or growth. A “cancer” or“malignancy” refers to a neoplasia or tumor that can invade adjacentspaces, tissues or organs. A “metastasis” refers to a neoplasia, tumor,cancer or malignancy that has disseminated or spread from its primarysite to one or more secondary sites, locations or regions within thesubject, in which the sites, locations or regions are distinct from theprimary tumor or cancer.

Neoplastic, tumor, cancer and malignant cells (metastatic ornon-metastatic) include dormant or residual neoplastic, tumor, cancerand malignant cells. Such cells typically consist of remnant tumor cellsthat are not dividing (G0-G1 arrest). These cells can persist in aprimary site or as disseminated neoplastic, tumor, cancer or malignantcells as a minimal residual disease. These dormant neoplastic, tumor,cancer or malignant cells remain unsymptomatic, but can develop severesymptoms and death once these dormant cells proliferate. Inventionmethods can be used to reduce or inhibit proliferation of dormantneoplastic, tumor, cancer or malignant cells, which can in turn inhibitor reduce tumor or cancer relapse, or tumor or cancer metastasis orprogression.

In accordance with the invention, methods of treating a subject having ametastatic or non-metastatic tumor, cancer, malignancy or neoplasia areprovided. In one embodiment, a method includes administering to asubject (in need of treatment) an amount of a fusion construct ofsufficient to treat (e.g., reduce or inhibit proliferation) themetastatic or non-metastatic tumor, cancer, malignancy or neoplasia.

The metastatic or non-metastatic tumor, cancer, malignancy or neoplasiamay be in any stage, e.g., early or advanced, such as a stage I, II,III, IV or V tumor. The metastatic or non-metastatic tumor, cancer,malignancy or neoplasia may have been subject to a prior treatment or bestabilized (non-progressing) or in remission.

In terms of metastasis, invention methods can be used to reduce orinhibit metastasis of a primary tumor or cancer to other sites, or theformation or establishment of metastatic tumors or cancers at othersites distal from the primary tumor or cancer thereby inhibiting orreducing tumor or cancer relapse or tumor or cancer progression. Thus,methods of the invention include, among other things, 1) reducing orinhibiting growth, proliferation, mobility or invasiveness of tumor orcancer cells that potentially or do develop metastases (e.g.,disseminated tumor cells, DTC); 2) reducing or inhibiting formation orestablishment of metastases arising from a primary tumor or cancer toone or more other sites, locations or regions distinct from the primarytumor or cancer; 3) reducing or inhibiting growth or proliferation of ametastasis at one or more other sites, locations or regions distinctfrom the primary tumor or cancer after a metastasis has formed or hasbeen established; and 4) reducing or inhibiting formation orestablishment of additional metastasis after the metastasis has beenformed or established.

Cells of a metastatic or non-metastatic tumor, cancer, malignancy orneoplasia may be aggregated in a “solid” cell mass or be dispersed ordiffused. A “solid” tumor refers to cancer, neoplasia or metastasis thattypically aggregates together and forms a mass. Specific non-limitingexamples include visceral tumors such as melanomas, breast, pancreatic,uterine and ovarian cancers, testicular cancer, including seminomas,gastric or colon cancer, hepatomas, adrenal, renal and bladdercarcinomas, lung, head and neck cancers and brain tumors/cancers.

Carcinomas, which refer to malignancies of epithelial or endocrinetissue, include respiratory system carcinomas, gastrointestinal systemcarcinomas, genitourinary system carcinomas, testicular carcinomas,breast carcinomas, prostatic carcinomas, endocrine system carcinomas,and melanomas.

Exemplary carcinomas include those forming from the uterus, cervix,lung, prostate, breast, head and neck, colon, pancreas, testes, adrenal,kidney, esophagus, stomach, liver and ovary. The term also includescarcinosarcomas, e.g., which include malignant tumors composed ofcarcinomatous and sarcomatous tissues. Adenocarcinoma includes acarcinoma of a glandular tissue, or in which the tumor forms a glandlike structure.

Sarcomas refer to malignant tumors of mesenchymal cell origin. Exemplarysarcomas include for example, lymphosarcoma, liposarcoma, osteosarcoma,chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma and fibrosarcoma.

Neural neoplasias include glioma, glioblastoma, meningioma,neuroblastoma, retinoblastoma, astrocytoma and oligodendrocytoma.

A “liquid tumor,” which refers to neoplasia that is dispersed or isdiffuse in nature, as they do not typically form a solid mass.Particular examples include neoplasia of the reticuloendothelial orhematopoietic system, such as lymphomas, myelomas and leukemias.Non-limiting examples of leukemias include acute and chroniclymphoblastic, myeolblastic and multiple myeloma. Typically, suchdiseases arise from poorly differentiated acute leukemias, e.g.,erythroblastic leukemia and acute megakaryoblastic leukemia. Specificmyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML). Lymphoid malignancies include, but are notlimited to, acute lymphoblastic leukemia (ALL), which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Specific malignant lymphomasinclude, non-Hodgkin lymphoma and variants, peripheral T cell lymphomas,adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL),large granular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

As disclosed herein, undesirable or aberrant cell proliferation orhyperproliferative disorders can occur in uterus, breast, vagina, cervixand fallopian tube. Endometriosis occurs when cells of the uterus growoutside of the uterus and into other areas, such as ovaries, bladder orbowel. Fibroids and polyps can affect uterus, breast, vagina, cervix andfallopian tube.

Thus, in accordance with the invention, there are provided methods oftreating endometriosis and fibroids or polyps. In one embodiment, amethod includes administering to a subject an amount of a fusionconstruct sufficient to treat endometriosis. In another embodiment, amethod includes administering to a subject an amount of a fusionconstruct sufficient to treat a fibroid or polyp.

Target cells include cells that participate in or a required forreproduction or fertility. Thus, in accordance with the invention, thereare provided methods of reducing fertility of an animal. In oneembodiment, a method includes administering to a subject an amount of afusion construct sufficient to reduce fertility or reduce the likelihoodof pregnancy or reducing sperm production in a male mammal.

As also disclosed herein, undesirable or aberrant cell proliferation orhyperproliferative disorders can occur in prostate. Thus, in accordancewith the invention, there are provided methods of treating benignprostate hyperplasia or metastatic prostate neoplasia. In oneembodiment, a method includes administering to a subject an amount of afusion construct sufficient to treat benign prostate hyperplasia ormetastatic prostate neoplasia.

Any composition, treatment, protocol, therapy or regimen having ananti-cell proliferative activity or effect can be combined with a fusionconstruct or used in combination in a method of the invention. Fusionconstructs and methods of the invention therefore includeanti-proliferative, anti-tumor, anti-cancer, anti-neoplastic andanti-metastatic treatments, protocols and therapies, which include anyother composition, treatment, protocol or therapeutic regimen thatinhibits, decreases, retards, slows, reduces or prevents ahyperproliferative disorder, such as tumor, cancer, malignant orneoplastic growth, progression, metastasis, proliferation or survival,or worsening in vitro or in vivo. Particular non-limiting examples of ananti-proliferative (e.g., tumor) therapy include chemotherapy,immunotherapy, radiotherapy (ionizing or chemical), local thermal(hyperthermia) therapy, surgical resection and vaccination. A fusionconstruct can be administered prior to, substantially contemporaneouslywith or following administration of the anti-cell proliferative,anti-neoplastic, anti-tumor, anti-cancer, anti-metastatic orimmune-enhancing treatment or therapy. A fusion construct can beadministered as a combination compositions with the anti-cellproliferative, anti-neoplastic, anti-tumor, anti-cancer, anti-metastaticor immune-enhancing treatment or therapy, metastatic or non-metastatictumor, cancer, malignancy or neoplasia.

Anti-proliferative, anti-neoplastic, anti-tumor, anti-cancer andanti-metastatic compositions, therapies, protocols or treatments includethose that prevent, disrupt, interrupt, inhibit or delay cell cycleprogression or cell proliferation; stimulate or enhance apoptosis orcell death, inhibit nucleic acid or protein synthesis or metabolism,inhibit cell division, or decrease, reduce or inhibit cell survival, orproduction or utilization of a necessary cell survival factor, growthfactor or signaling pathway (extracellular or intracellular).Non-limiting examples of chemical agent classes having anti-cellproliferative, anti-neoplastic, anti-tumor, anti-cancer andanti-metastatic activities include alkylating agents, anti-metabolites,plant extracts, plant alkaloids, nitrosoureas, hormones, nucleoside andnucleotide analogues. Specific examples of drugs having anti-cellproliferative, anti-neoplastic, anti-tumor, anti-cancer andanti-metastatic activities include cyclophosphamide, azathioprine,cyclosporin A, prednisolone, melphalan, chlorambucil, mechlorethamine,busulphan, methotrexate, 6-mercaptopurine, thioguanine, 5-fluorouracil,cytosine arabinoside, AZT, 5-azacytidine (5-AZC) and 5-azacytidinerelated compounds such as decitabine (5-aza-2′deoxycytidine),cytarabine, 1-beta-D-arabinofuranosyl-5-azacytosine anddihydro-5-azacytidine, bleomycin, actinomycin D, mithramycin, mitomycinC, carmustine, lomustine, semustine, streptozotocin, hydroxyurea,cisplatin, mitotane, procarbazine, dacarbazine, taxol, vinblastine,vincristine, doxorubicin and dibromomannitol etc.

Additional agents that are applicable with fusion constructs and methodsare known in the art and can be employed. For example, biologicals suchas antibodies, cell growth factors, cell survival factors, celldifferentiative factors, cytokines and chemokines can be administered.Non-limiting examples of monoclonal antibodies include rituximab(Rituxan®), trastuzumab (Herceptin), bevacizumab (Avastin), cetuximab(Erbitux), alemtuzumab (Campath), panitumumab (Vectibix), ibritumomabtiuxetan (Zevalin), tositumomab (Bexxar) etc. which can be used incombination with, inter alia, a fusion construct in accordance with theinvention. Other targeted drugs that are applicable for use with thefusion constructs are imatinib (Gleevec), gefitinib (Iressa), bortzomib(Velcade), lapatinib (Tykerb), sunitinib (Sutent), sorafenib (Nevaxar),nilotinib (Tasigna) etc Non-limiting examples of cell growth factors,cell survival factors, cell differentiative factors, cytokines andchemokines include IL-2, IL-1α, IL-β, IL-3, IL-6, IL-7,granulocyte-macrophage-colony stimulating factor (GMCSF), IFN-γ, IL-12,TNF-α, TNFβ, MIP-1α, MIP-1β, RANTES, SDF-1, MCP-1, MCP-2, MCP-3, MCP-4,eotaxin, eotaxin-2, I-309/TCA3, ATAC, HCC-1, HCC-2, HCC-3, LARC/MIP-3α,PARC, TARC, CKβ, CKβ6, CKβ7, CKβ8, CKβ9, CKβ11, CKβ12, C10, IL-8, GROα,GROβ, ENA-78, GCP-2, PBP/CTAPIIIβ-TG/NAP-2, Mig, PBSF/SDF-1 andlymphotactin.

Additional non-limiting examples include immune-enhancing treatments andtherapies, which include cell based therapies. In particular,immune-enhancing treatments and therapies include administeringlymphocytes, plasma cells, macrophages, dendritic cells, NK cells andB-cells.

Methods of treating a metastatic or non-metastatic tumor, cancer,malignancy or neoplasia, methods of treating a subject in need oftreatment due to having or at risk of having a metastatic ornon-metastatic tumor, cancer, malignancy or neoplasia, and methods ofincreasing effectiveness or improving an anti-proliferative, anti-tumor,anti-cancer, anti-neoplasia or anti-malignancy, therapy are provided. Inrespective embodiments, a method includes administering to a subjectwith or at risk of a metastatic or non-metastatic tumor, cancer,malignancy or neoplasia, an amount of a fusion construct sufficient totreat the metastatic or non-metastatic tumor, cancer, malignancy orneoplasia; administering to the subject an amount of a fusion constructsufficient to treat the subject; and administering to a subject that isundergoing or has undergone metastatic or non-metastatic tumor, cancer,malignancy or neoplasia therapy, an amount of a fusion constructsufficient to increase effectiveness of the anti-proliferative,anti-tumor, anti-cancer, anti-neoplasia or anti-malignancy therapy.

Methods of the invention may be practiced prior to (i.e. prophylaxis),concurrently with or after evidence of the presence of undesirable oraberrant cell proliferation or a hyperproliferative disorder, disease orcondition begins (e.g., one or more symptoms). Administering a fusionconstruct prior to, concurrently with or immediately followingdevelopment of a symptom of undesirable or aberrant cell proliferationor a hyperproliferative disorder may decrease the occurrence, frequency,severity, progression, or duration of one or more symptoms of theundesirable or aberrant cell proliferation or a hyperproliferativedisorder, disease or condition in the subject. In addition,administering a fusion construct prior to, concurrently with orimmediately following development of one or more symptoms of theundesirable or aberrant cell proliferation or a hyperproliferativedisorder, disease or condition may inhibit, decrease or prevent thespread or dissemination of hyperproliferating cells (e.g., metastasis)to other sites, regions, tissues or organs in a subject, orestablishment of hyperproliferating cells (e.g., metastasis) at othersites, regions, tissues or organs in a subject.

Fusion constructs and the methods of the invention, such as treatmentmethods, can provide a detectable or measurable therapeutic benefit orimprovement to a subject. A therapeutic benefit or improvement is anymeasurable or detectable, objective or subjective, transient, temporary,or longer-term benefit to the subject or improvement in the condition,disorder or disease, an adverse symptom, consequence or underlyingcause, of any degree, in a tissue, organ, cell or cell population of thesubject. Therapeutic benefits and improvements include, but are notlimited to, reducing or decreasing occurrence, frequency, severity,progression, or duration of one or more symptoms or complicationsassociated with a disorder, disease or condition, or an underlying causeor consequential effect of the disorder, disease or condition. Fusionconstructs and methods of the invention therefore include providing atherapeutic benefit or improvement to a subject.

In a method of the invention in which a therapeutic benefit orimprovement is a desired outcome, a fusion construct of the inventioncan be administered in a sufficient or effective amount to a subject inneed thereof. An “amount sufficient” or “amount effective” refers to anamount that provides, in single or multiple doses, alone or incombination, with one or more other compositions (therapeutic agentssuch as a chemotheraputic or immune stimulating drug), treatments,protocols, or therapeutic regimens agents, a detectable response of anyduration of time (long or short term), a desired outcome in or a benefitto a subject of any measurable or detectable degree or for any durationof time (e.g., for hours, days, months, years, or cured). The doses or“sufficient amount” or “effective amount” for treatment (e.g., toprovide a therapeutic benefit or improvement) typically are effective toameliorate a disorder, disease or condition, or one, multiple or alladverse symptoms, consequences or complications of the disorder, diseaseor condition, to a measurable extent, although reducing or inhibiting aprogression or worsening of the disorder, disease or condition or asymptom, is considered a satisfactory outcome.

The term “ameliorate” means a detectable objective or subjectiveimprovement in a subject's condition. A detectable improvement includesa subjective or objective reduction in the occurrence, frequency,severity, progression, or duration of a symptom caused by or associatedwith a disorder, disease or condition, an improvement in an underlyingcause or a consequence of the disorder, disease or condition, or areversal of the disorder, disease or condition.

Treatment can therefore result in inhibiting, reducing or preventing adisorder, disease or condition, or an associated symptom or consequence,or underlying cause; inhibiting, reducing or preventing a progression orworsening of a disorder, disease, condition, symptom or consequence, orunderlying cause; or further deterioration or occurrence of one or moreadditional symptoms of the disorder, disease condition, or symptom.Thus, a successful treatment outcome leads to a “therapeutic effect,” or“benefit” or inhibiting, reducing or preventing the occurrence,frequency, severity, progression, or duration of one or more symptoms orunderlying causes or consequences of a condition, disorder, disease orsymptom in the subject. Treatment methods affecting one or moreunderlying causes of the condition, disorder, disease or symptom aretherefore considered to be beneficial. Stabilizing or inhibitingprogression or worsening of a disorder or condition is also a successfultreatment outcome.

A therapeutic benefit or improvement therefore need not be completeablation of any one, most or all symptoms, complications, consequencesor underlying causes associated with the condition, disorder or disease.Thus, a satisfactory endpoint is achieved when there is an incrementalimprovement in a subject's condition, or a partial reduction in theoccurrence, frequency, severity, progression, or duration, or inhibitionor reversal, of one or more associated adverse symptoms or complicationsor consequences or underlying causes, worsening or progression (e.g.,stabilizing one or more symptoms or complications of the condition,disorder or disease), of one or more of the physiological, biochemicalor cellular manifestations or characteristics of the disorder ordisease, over a short or long duration of time (hours, days, weeks,months, etc.).

In particular embodiments, a method of treatment results in partial orcomplete destruction of a metastatic or non-metastatic tumor, cancer,malignant or neoplastic cell mass, volume, size or numbers of cells;results in stimulating, inducing or increasing metastatic ornon-metastatic tumor, cancer, malignant or neoplastic cell necrosis,lysis or apoptosis; results in reducing metastatic or non-metastatictumor, cancer, malignant or neoplastic volume, size, cell mass; resultsin inhibiting or preventing progression or an increase in metastatic ornon-metastatic tumor, cancer, malignant or neoplastic volume, mass, sizeor cell numbers; results in inhibiting or decreasing the spread ordissemination of hyperproliferating cells (e.g., metastasis) to other(secondary) sites, regions, tissues or organs in a subject, orestablishment of hyperproliferating cells (e.g., metastasis) at other(secondary) sites, regions, tissues or organs in a subject; or resultsin prolonging lifespan of the subject. In additional particularembodiments, a method of treatment results in reducing or decreasingseverity, duration or frequency of an adverse symptom or complicationassociated with or caused by the metastatic or non-metastatic tumor,cancer, malignancy or neoplasia.

An amount sufficient or an amount effective can but need not be providedin a single administration and, can but need not be, administered aloneor in combination with another composition (e.g., chemotherapeutic orimmune enhancing or stimulating agent), treatment, protocol ortherapeutic regimen. For example, the amount may be proportionallyincreased as indicated by the need of the subject, status of thedisorder, disease or condition treated or the side effects of treatment.In addition, an amount sufficient or an amount effective need not besufficient or effective if given in single or multiple doses without asecond composition (e.g., chemotherapeutic or immune stimulating agent),treatment, protocol or therapeutic regimen, since additional doses,amounts or duration above and beyond such doses, or additionalcompositions (e.g., chemotherapeutic or immune stimulating agents),treatments, protocols or therapeutic regimens may be included in orderto be considered effective or sufficient in a given subject. Amountsconsidered sufficient also include amounts that result in a reduction ofthe use of another treatment, therapeutic regimen or protocol.

An amount sufficient or an amount effective need not be effective ineach and every subject treated, prophylactically or therapeutically, nora majority of treated subjects in a given group or population. As istypical for treatment or therapeutic methods, some subjects will exhibitgreater or less response to a given treatment, therapeutic regimen orprotocol. An amount sufficient or an amount effective refers tosufficiency or effectiveness in a particular subject, not a group or thegeneral population. Such amounts will depend in part upon the conditiontreated, such as the type or stage of undesirable or aberrant cellproliferation or hyperproliferative disorder (e.g., a metastatic ornon-metastatic tumor, cancer, malignancy or neoplasia), the therapeuticeffect desired, as well as the individual subject (e.g., thebioavailability within the subject, gender, age, etc.).

Particular non-limiting examples of therapeutic benefit or improvementfor undesirable or aberrant cell proliferation, such as ahyperproliferative disorder (e.g., a metastatic or non-metastatic tumor,cancer, malignancy or neoplasia) include a reduction in cell size, massor volume, inhibiting an increase in cell size, mass or volume, aslowing or inhibition of worsening or progression, stimulating cellnecrosis, lysis or apoptosis, reducing or inhibiting neoplastic or tumormalignancy or metastasis, reducing mortality, and prolonging lifespan ofa subject. Thus, inhibiting or delaying an increase in cell size, mass,volume or metastasis (stabilization) can increase lifespan (reducemortality) even if only for a few days, weeks or months, even thoughcomplete ablation of the metastatic or non-metastatic tumor, cancer,malignancy or neoplasia has not occurred. Adverse symptoms andcomplications associated with a hyperproliferative disorder (e.g., ametastatic or non-metastatic tumor, cancer, malignancy or neoplasia)that can be reduced or decreased include, for example, pain, nausea,discomfort, lack of appetite, lethargy and weakness. A reduction in theoccurrence, frequency, severity, progression, or duration of a symptomof undesirable or aberrant cell proliferation, such as ahyperproliferative disorder (e.g., a metastatic or non-metastatic tumor,cancer, malignancy or neoplasia), such as an improvement in subjectivefeeling (e.g., increased energy, appetite, reduced nausea, improvedmobility or psychological well being, etc.), are therefore all examplesof therapeutic benefit or improvement.

For example, a sufficient or effective amount of a fusion construct isconsidered as having a therapeutic effect if administration results inless chemotherapeutic drug, radiation or immunotherapy being requiredfor treatment of undesirable or aberrant cell proliferation, such as ahyperproliferative disorder (e.g., a metastatic or non-metastatic tumor,cancer, malignancy or neoplasia).

The term “subject” refers to animals, typically mammalian animals, suchas humans, non human primates (apes, gibbons, chimpanzees, orangutans,macaques), domestic animals (dogs and cats), farm animals (horses, cows,goats, sheep, pigs) and experimental animal (mouse, rat, rabbit, guineapig). Subjects include animal disease models, for example, animal modelsof undesirable or aberrant cell proliferation, such as ahyperproliferative disorder (e.g., a metastatic or non-metastatic tumor,cancer, malignancy or neoplasia) for analysis of fusion constructs invivo.

Subjects appropriate for treatment include those having or at risk ofhaving a metastatic or non-metastatic tumor, cancer, malignant orneoplastic cell, those undergoing as well as those who are undergoing orhave undergone anti-proliferative (e.g., metastatic or non-metastatictumor, cancer, malignancy or neoplasia) therapy, including subjectswhere the tumor is in remission. “At risk” subjects typically have riskfactors associated with undesirable or aberrant cell proliferation,development of hyperplasia (e.g., a tumor).

Particular examples of at risk or candidate subjects include those withcells that express a receptor, ligand, antigen or antibody to which afusion construct can bind, particularly where cells targeted fornecrosis, lysis, killing or destruction express greater numbers oramounts of receptor, ligand, antigen or antibody than non-target cells.Such cells can be selectively or preferentially targeted for necrosis,lysis or killing.

At risk subjects also include those that are candidates for and thosethat have undergone surgical resection, chemotherapy, immunotherapy,ionizing or chemical radiotherapy, local or regional thermal(hyperthermia) therapy, or vaccination. The invention is thereforeapplicable to treating a subject who is at risk of a metastatic ornon-metastatic tumor, cancer, malignancy or neoplasia or a complicationassociated with a metastatic or non-metastatic tumor, cancer, malignancyor neoplasia, for example, due to metastatic or non-metastatic tumor,cancer, malignancy or neoplasia reappearance or regrowth following aperiod of stability or remission.

Risk factors include gender, lifestyle (diet, smoking), occupation(medical and clinical personnel, agricultural and livestock workers),environmental factors (carcinogen exposure), family history (autoimmunedisorders, diabetes, etc.), genetic predisposition, etc. For example,subjects at risk for developing melanoma include excess sun exposure(ultraviolet radiation), fair skin, high numbers of naevi (dysplasticnevus), patient phenotype, family history, or a history of a previousmelanoma. Subjects at risk for developing cancer can therefore beidentified by lifestyle, occupation, environmental factors, familyhistory, and genetic screens for tumor associated genes, gene deletionsor gene mutations. Subjects at risk for developing breast cancer lackBrca1, for example. Subjects at risk for developing colon cancer haveearly age or high frequency polyp formation, or deleted or mutated tumorsuppressor genes, such as adenomatous polyposis coli (APC), for example.

Subjects also include those precluded from other treatments. Forexample, certain subjects may not be good candidates for surgicalresection, chemotherapy, immunotherapy, ionizing or chemicalradiotherapy, local or regional thermal (hyperthermia) therapy, orvaccination. Thus, candidate subjects for treatment in accordance withthe invention include those that are not a candidate for surgicalresection, chemotherapy, immunotherapy, ionizing or chemicalradiotherapy, local or regional thermal (hyperthermia) therapy, orvaccination.

Fusion constructs may be formulated in a unit dose or unit dosage form.In a particular embodiment, a fusion construct is in an amount effectiveto treat a subject having undesirable or aberrant cell proliferation ora hyperproliferative disorder. In an additional particular embodiment, afusion construct is in an amount effective to treat a subject having ametastatic or non-metastatic tumor, cancer, malignancy or neoplasia. Ina further particular embodiment, a fusion construct is in an amounteffective to reduce fertility of a subject. Exemplary unit doses rangefrom about 25-250, 250-500, 500-1000, 1000-2500 or 2500-5000,5000-25,000, 5000-50,000 ng; and from about 25-250, 250-500, 500-1000,1000-2500 or 2500-5000, 5000-25,000, 5000-50,000 μg.

Compositions and methods of the invention may be contacted or providedin vitro, ex vivo or in vivo. Compositions can be administered toprovide the intended effect as a single or multiple dosages, forexample, in an effective or sufficient amount. Exemplary doses rangefrom about 25-250, 250-500, 500-1000, 1000-2500 or 2500-5000,5000-25,000, 5000-50,000 pg/kg; from about 50-500, 500-5000, 5000-25,000or 25,000-50,000 ng/kg; and from about 25-250, 250-500, 500-1000,1000-2500 or 2500-5000, 5000-25,000, 5000-50,000 μg/kg, on consecutivedays, or alternating days or intermittently. Single or multiple dosescan be administered on consecutive days, alternating days orintermittently.

Compositions can be administered and methods may be practiced viasystemic, regional or local administration, by any route. For example, afusion construct can be administered systemically, regionally orlocally, intravenously, orally (e.g., ingestion or inhalation),intramuscularly, intraperitoneally, intradermally, subcutaneously,intracavity, intracranially, transdermally (topical), parenterally, e.g.transmucosally or rectally. Compositions and methods of the inventionincluding pharmaceutical formulations can be administered via a(micro)encapsulated delivery system or packaged into an implant foradministration.

The invention further provides fusion constructs and methods wherein thefusion constructs are included in pharmaceutical compositions. Apharmaceutical composition refers to “pharmaceutically acceptable” and“physiologically acceptable” carriers, diluents or excipients. As usedherein, the term “pharmaceutically acceptable” and “physiologicallyacceptable,” when referring to carriers, diluents or excipients includessolvents (aqueous or non-aqueous), detergents, solutions, emulsions,dispersion media, coatings, isotonic and absorption promoting ordelaying agents, compatible with pharmaceutical administration and withthe other components of the formulation. Such formulations can becontained in a tablet (coated or uncoated), capsule (hard or soft),microbead, emulsion, powder, granule, crystal, suspension, syrup orelixir.

Pharmaceutical compositions can be formulated to be compatible with aparticular route of administration. Compositions for parenteral,intradermal, or subcutaneous administration can include a sterilediluent, such as water, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents. Thepreparation may contain one or more preservatives to preventmicroorganism growth (e.g., antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose).

Pharmaceutical compositions for injection include sterile aqueoussolutions (where water soluble) or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, Cremophor EL™ (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and polyetheylene glycol), andsuitable mixtures thereof. Fluidity can be maintained, for example, bythe use of a coating such as lecithin, or by the use of surfactants.Antibacterial and antifungal agents include, for example, parabens,chlorobutanol, phenol, ascorbic acid and thimerosal. Including an agentthat delays absorption, for example, aluminum monostearate and gelatincan prolonged absorption of injectable compositions.

Additional pharmaceutical formulations and delivery systems are known inthe art and are applicable in the methods of the invention (see, e.g.,Remington's Pharmaceutical Sciences (1990) 18th ed., Mack PublishingCo., Easton, Pa.; The Merck Index (1996) 12th ed., Merck PublishingGroup, Whitehouse, N.J.; Pharmaceutical Principles of Solid DosageForms, Technonic Publishing Co., Inc., Lancaster, Pa., (1993); andPoznansky, et al., Drug Delivery Systems, R. L. Juliano, ed., Oxford,N.Y. (1980), pp. 253-315).

The invention provides kits including fusion constructs of theinvention, combination compositions and pharmaceutical formulationsthereof, packaged into suitable packaging material. A kit optionallyincludes a label or packaging insert including a description of thecomponents or instructions for use in vitro, in vivo, or ex vivo, of thecomponents therein. Exemplary instructions include instructions forreducing or inhibiting proliferation of a cell, reducing or inhibitingproliferation of undesirable or aberrant cells, such as ahyperproliferating cell, reducing or inhibiting proliferation of ametastatic or non-metastatic tumor, cancer, malignant or neoplasticcell, treating a subject having a hyperproliferative disorder, treatinga subject having a metastatic or non-metastatic tumor, cancer,malignancy or neoplasia, or reducing fertility of an animal.

A kit can contain a collection of such components, e.g., two or morefusion constructs alone, or in combination with another therapeuticallyuseful composition (e.g., an anti-proliferative or immune-enhancingdrug).

The term “packaging material” refers to a physical structure housing thecomponents of the kit. The packaging material can maintain thecomponents sterilely, and can be made of material commonly used for suchpurposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules,vials, tubes, etc.).

Kits of the invention can include labels or inserts. Labels or insertsinclude “printed matter,” e.g., paper or cardboard, or separate oraffixed to a component, a kit or packing material (e.g., a box), orattached to an ampule, tube or vial containing a kit component. Labelsor inserts can additionally include a computer readable medium, such asa disk (e.g., floppy diskette, hard disk, ZIP disk), optical disk suchas CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storagemedia such as RAM and ROM or hybrids of these such as magnetic/opticalstorage media, FLASH media or memory type cards.

Labels or inserts can include identifying information of one or morecomponents therein, dose amounts, clinical pharmacology of the activeingredient(s) including mechanism of action, pharmacokinetics andpharmacodynamics. Labels or inserts can include information identifyingmanufacturer information, lot numbers, manufacturer location and date.

Labels or inserts can include information on a condition, disorder,disease or symptom for which a kit component may be used. Labels orinserts can include instructions for the clinician or for a subject forusing one or more of the kit components in a method, treatment protocolor therapeutic regimen. Instructions can include dosage amounts,frequency or duration, and instructions for practicing any of themethods, treatment protocols or therapeutic regimes set forth herein.Exemplary instructions include, instructions for treating an undesirableor aberrant cell proliferation, hyperproliferating cells and disorders(e.g., metastatic or non-metastatic tumor, cancer, malignancy orneoplasia). Kits of the invention therefore can additionally includelabels or instructions for practicing any of the methods of theinvention described herein including treatment methods.

Labels or inserts can include information on any benefit that acomponent may provide, such as a prophylactic or therapeutic benefit.Labels or inserts can include information on potential adverse sideeffects, such as warnings to the subject or clinician regardingsituations where it would not be appropriate to use a particularcomposition. Adverse side effects could also occur when the subject has,will be or is currently taking one or more other medications that may beincompatible with the composition, or the subject has, will be or iscurrently undergoing another treatment protocol or therapeutic regimenwhich would be incompatible with the composition and, therefore,instructions could include information regarding such incompatibilities.

Invention kits can additionally include other components. Each componentof the kit can be enclosed within an individual container and all of thevarious containers can be within a single package. Invention kits can bedesigned for cold storage. Invention kits can further be designed tocontain host cells expressing fusion constructs of the invention, orthat contain nucleic acids encoding fusion constructs. The cells in thekit can be maintained under appropriate storage conditions until thecells are ready to be used. For example, a kit including one or morecells can contain appropriate cell storage medium so that the cells canbe thawed and grown.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described herein.

All applications, publications, patents and other references, GenBankcitations and ATCC citations cited herein are incorporated by referencein their entirety. In case of conflict, the specification, includingdefinitions, will control.

As used herein, the singular forms “a”, “and,” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “a fusion construct” or a “lytic domain” includesa plurality of such fusion constructs or lytic domains, and so forth.

As used herein, all numerical values or numerical ranges includeintegers within such ranges and fractions of the values or the integerswithin ranges unless the context clearly indicates otherwise. Thus, forexample, reference to a range of 90-100%, includes 91%, 92%, 93%, 94%,95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc.,92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth.

The invention is generally disclosed herein using affirmative languageto describe the numerous embodiments. The invention also specificallyincludes embodiments in which particular subject matter is excluded, infull or in part, such as substances or materials, method steps andconditions, protocols, procedures, assays or analysis. Thus, even thoughthe invention is generally not expressed herein in terms of what theinvention does not include aspects that are not expressly included inthe invention are nevertheless disclosed herein.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, the following examples are intended to illustrate but notlimit the scope of invention described in the claims.

The Sequence Listing submitted herewith in computer readable form isincorporated herein by reference.

EXAMPLES Example 1

Initial studies included in vitro screening of 28 lytic domain peptidesthat contained different hinge sequences between lytic peptide moietyand ligands; that contained 30% of D amino acids (D-enantiomers), thatwere 18 and 15 amino acids in length for the lytic peptide moiety andcontained hinge sequence or their D-enantiomers. The introduction ofα-aminocaproic acid as a spacer on 21, 18 and 15 amino acid Phor21analogs was studied. The ligands chosen for study included βCG-ala, a 15amino acid fragment of the binding moiety of the beta chain fromchorionic gonadotropin, and LHRH, a decapeptide that represents a fullfunctioning ligand.

Example 2

This example describes screening for cell toxicity (IC₅₀) and hemolyticactivity using a human breast cancer cell line.

Eighteen βCG-ala and eight LHRH conjugated fusion constructs werestudied and compared to Phor21-βCG-ala and unconjugated Phor21 andPhor18 (338913)=CLIP71 peptides. The human breast cancer cell lineMDA-MB-435S.luc, which over expresses chorionic gonadotrophin (CG) andluteinizing hormone-releasing hormone (LHRH) receptors, was used toscreen at passage numbers 248-252. The MDA-MB-435S.luc cell line wasconstructed from the MDA-MB-435S cell line obtained from the AmericanType Cell Culture Collection by stable transfection with the plasmidPRC/CMV-luc containing the Photinus pyralis luciferase gene and anantibody resistance gene by lipofection. The stably transfected cellline was selected using G418 and the clones with the highest expressionfor the luciferase gene were tested for their LH and LHRH receptorexpression.

MDA-MB-435S.luc cells were grown in Leibovitz's L 15 medium, 10% fetalbovine serum, 0.01 mg/ml bovine insulin, 100 IU/ml penicillin, 100microg/ml streptomycin. The cells were cultured in tight closed flasks.The incubations were conducted using 96 well plates at 10,000 cells perwell. Cells were typically seeded into 96 well plates and media wasreplaced after 48 hours of incubation. Each assay was conducted atincreasing concentrations of 0, 0.001, 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10and 100 micromolar doses of lytic peptide-binding domain conjugate. Eachlytic peptide-binding domain conjugate provided in lyophilized form andwas freshly dissolved in saline and added to cells. The duration ofincubation was typically 24 h, and cell viability assays were conductedusing formazan conversion assays (MTT assay). Controls contained salineor 0.1% triton as reference for 0 and 100% cell death, respectively.

Data were processed and analyzed using Graph Pad Prizm 4™ software(Graph Pad Prizm, Inc). Statistical analysis for significance wasdetermined by a two-tailed Student's T-test. Each study was conducted toachieve an N of at least 8.

The effect of increasing the length of the fusion construct wasascertained (Javadpour et al., J Med Chem 39:3107 (1996); Javadpour andBarkeley, Biochemistry 36:9540 (1997); Leuschner and Hansel, CurrentPharmaceutical Design, 10:2299 (2004); and Leuschner and Hansel, BiolReprod 73:255 (2005)). Lytic peptides conjugated at the C-terminus toβCG-ala showed increasing toxicity with increasing length of theconstruct. The IC₅₀ for peptides of various lengths were: 14 amino acids(Phor 14) 5.74, 15 amino acids (Phor15) 1.92, 18 amino acids(Phor18=CLIP71) 1.09, 21 amino acids (Phor21) 2.31 and 28 amino acids(Phor28) 1.36 μM (Table 3).

The effect of the position of the binding moiety (N- or C-terminus) wasascertained. In brief, Phor21-βCG-ala (C-terminus), βCG-ala Phor21(N-terminus), LHRH-Phor21 (N-terminus) and Phor21-LHRH (C-terminus)fusion constructs were studied. The IC₅₀ of the peptides were:Phor21-βCG-ala 2.3 μM, for βCG-ala-Phor21 4.7 μM, for LHRH-Phor21 2.65μM, and for Phor21-LHRH 1.71 μM. The data demonstrate that C-terminalpositioning of βCG-ala and LHRH binding moieties showed greater toxicitythan if the binding moiety was positioned at the N-terminus.

LHRH-receptor is present in many human cancers (see Table 1). Theactivity of LHRH as the binding moiety was compared to βCG-ala as thebinding moiety.

Toxicities of LHRH-Phor21 and Phor21-βCG-ala were compared in humanMDA-MB-435S.luc breast cancer cells in vitro, at 2 or 24 hourincubation. The data indicate that LHRH-Phor21 killed cells faster thanPhor21-βCG-ala, LHRH-Phor21 eliciting cell killing within 2 hours (FIG.1).

TABLE 1 LH and LHRH Receptors in Human Cancers Cancer Type LH ReceptorsLHRH-Receptors Breast  72% 52% Prostate 100% 86% Ovarian  40% 80%Endometrial  17% 80% Pancreatic N.D. 68% Lung Yes N.D. Melanoma  68% YesBrain N.D. Yes Colon N.D. Yes Oral N.D. Yes

Introduction of hinge, spacer or linker sequences between the lyticdomain and binding moiety resulted in peptides with greater potency thanPhor21-βCG-ala in cell killing. (FIG. 2, Table 2). Whereas introductionof hinge sequences or spacers in the Phor21-βCG-ala fusion construct didnot change cell killing activity significantly, the effect of ASAAS(SEQ. ID NO. 11) as a hinge sequence significantly increased thetoxicity of lytic peptides with 15 amino acids for the βCG-conjugate;this effect was absent in the case of the βCG and LHRH conjugatedpeptide Phor18-LHRH and Phor18-ASAAS-LHRH, which were equally effectivein vitro (Table 2, FIG. 4). A similar effect was observed when the hingesequence was substituted by a 6 carbon spacer, alpha amino caproic acid(Table 2). The substitution of alanine by glycine in the second hingesequence (GSGGS (SEQ. ID NO. 10)) resulted in significantly loweractivity, suggesting that glycine may have had a helix destabilizingeffect. (FIG. 2, Table 2).

TABLE 2 Effect of peptide length of βCG-ala conjugated peptides NoneGSGGS ASAAS Amino-caproic Peptide [IC₅₀ μM] [IC₅₀ μM] [IC₅₀ μM] acid[IC₅₀ μM] Phor21 2.3 3.27 2.77 2.75 Phor18 1.09* 2.32 2.07 NA Phor151.92 2.96 1.48* 1.31* Phor14 6.72 NA NA NA LHRH-conjugated peptidesPhor21 1.31 NA Phor18 0.87 0.89

To ascertain the effect of D-amino acid substitutions, a fusionconstruct Phor21-βCG-ala synthesized as D enantiomer (hereinafterreferred to as D-ala-Phor21-βCG-ala). This fusion construct showedcomparable toxicity to MDA-MB-435S.luc breast cancer cells in vitro(Phor21-βCG-ala 2.31 μM, D-ala-Phor21-βCG-ala 2.15 μM (Table 3);D-ala-Phor18-βCG-ala was 1.4 fold more potent than Phor21-βCG-ala (IC₅₀1.6 μM), the LHRH counterpart was significantly more potent asD-enantiomer (Phor21-LHRH (IC₅₀ of 1.31 μM) compared toD-ala-Phor21-LHRH with IC₅₀ of 0.75 μM, D-Phor18-LHRH with IC₅₀ of 1.42μM and Phor18-Lupron with IC₅₀ of 1.95 μM.

The IC_(50-values) for βCG-ala and LHRH-conjugated lytic peptides inMDA-MB-435S.luc breast cancer cells are summarized in Table 3 and FIG.3. In brief, peptides with significantly lower IC₅₀ than Phor21-βCG-ala(2.31±0.16) were: Phor28-βCG-ala (1.36±0.09 μM; p<0.0001),Phor15-ASAAS-βCG-ala (1.48±0.24 μM; p<0.005), Phor15-C₆-βCG-ala(1.31±0.17 μM; p<0.004) (C₆=6 aminocaproic acid), Phor18-βCG-ala(1.09±0.17 μM; p<0.0001), Phor21-LHRH (1.31±0.1 μM; p<0.0001),D-ala-Phor21-LHRH (0.75±0.1 μM; p<0.0001), Phor18-ASAAS-LHRH (0.88±0.12μM; p<0.0001), Phor18-LHRH (0.87±0.11 μM; p<0.0001), (KKKFAFA)₃ (SEQ. IDNO. 16)-LHRH (0.78±0.21 μM; p<0.0004), and D-ala-Phor18-LHRH (1.42±0.08μM; p<0.004).

LHRH fusion constructs were in general more potent (more toxic andfaster acting, FIG. 1) than βCG-ala fusion constructs. In brief,Phor21-LHRH, D-ala-Phor21-LHRH, Phor18-ASAAS-LHRH, Phor18-LHRH andpeptide control 338614 ((KKKFAFA)₃ (SEQ. ID NO. 16)=inactive peptide)were significantly more toxic to human breast cancer cells compared toPhor21-βCG-ala (p<0.003). All LHRH fusion constructs were about equallyeffective, except for LHRH-Phor21 which was significantly less potentwhen the binding moiety was positioned at the C terminus relative to thelytic portion. D-ala-Phor18-LHRH was equally effective compared toPhor21-LHRH; Phor18-Lupron were less toxic, but comparable toPhor21-βCG-ala. (FIG. 4, Table 3; Lupron is QHWSY(D-Leu)LRPNEt).

Fusion constructs with significantly lower IC_(50-values) thanPhor21-LHRH (1.34±0.1 μM) were: D-ala-Phor21-LHRH (0.75±0.12 μM;p<0.002), Phor18-ASAAS-LHRH (0.88±0.11 μM; p<0.0001), Phor18-LHRH(0.87±0.12 μM; p<0.004), and (KKKFAFA)₃ (SEQ. ID NO. 16)-LHRH (0.78±0.21μM; p<0.04). When the same fusion constructs were compared betweenβCG-ala and LHRH binding moieties, in all cases LHRH fusion constructswere significantly more toxic compared to their βCG-ala counterparts.

TABLE 3 Cytolytic peptides and peptide conjugates—summary of IC₅₀ andHA₅₀ characteristics in MDA-MB-435S.luc cells Peptide content Peptide IDDescription [%] IC₅₀ [μM] HA₅₀ [μM] IC₅₀/HA₅₀ 337464 Phor14-βCG-ala 84.96.74 ± 1.7 *** 1203 ± 586  0.005 323033 Phor21-βCG-ala 85.3 2.31 ± 0.1673.44 ± 6.9  0.03 337465 Phor28-βCG-ala 85.1 1.36 ± 0.09 *** 337466Phor21-GSGGS- 84.6 3.27 ± 0.45 153.6 ± 25   0.02 βCG-ala 337467Phor18-GSGGS- 87.6 2.32 ± 0.44 723.6 ± 219   0.003 βCG-ala 337468Phor15-GSGGS- 85.7 2.96 ± 0.43 βCG-ala 337469 Phor21-ASAAS- 83.5 2.77 ±0.18  72 ± 8.9 0.037 βCG-ala 337470 Phor18-ASAAS- 86.6 2.07 ± 0.2 578 ±241 0.0036 βCG-ala 337471 Phor15-ASAAS- 86.2 1.48 ± 0.24 ** 421 ± 1100.0035 βCG-ala 337472 Phor21-C₆-βCG-ala 85 2.25 ± 0.3 337473Phor18-C₆-βCG-ala 84.3  3.9 ± 0.9 337474 Phor15-C₆-βCG-ala 87.5 1.31 ±0.17 *** Not lytic 0 323033 Phor21-βCG-ala 85.3 2.31 ± 0.16 337476Phor18-βCG-ala 84.1 1.09 ± 0.17 *** 169.8 ± 24   0.006 337477Phor15-βCG-ala 85.9 1.92 ± 0.43 337478 βCG-ala-Phor21 84 4.75 ± 1.1337479 LHRH-Phor21 87.3 2.65 ± 0.12  33 ± 3.6 0.08 337480 Phor21-LHRH82.4 1.31 ± 0.1 ***  25 ± 4.3 0.07 337481 (D Ala)-Phor21- 82.7 2.15 ±0.26 Not lytic 0 βCG-ala 338982 Phor21 77.9  1.5 ± 0.2 Not lytic 0 N = 8338983 Phor 18 75.3 2.06 ± 0.5 Not lytic 0 N = 16 338984 (KKKFAFA)₃-83.8  4.4 ± 2.0 Not lytic 0 βCG-ala N = 29 338611 (D Ala)-Phor21- 85.10.75 ± 0.1 672.9 ± 155   0.001 LHRH N = 8 ** 338612 Phor18-ASAAS- 78.90.88 ± 0.12 410.1 ± 42   0.002 LHRH N = 36 *** 338613 Phor18-LHRH 81.40.87 ± 0.11 297.9 ± 35   0.003 N = 17 ** 338614 (KKKFAFA)₃- 81.4 0.78 ±0.21 95.7 ± 46.7 0.008 LHRH N = 12 *** 337476 Phor18-βCG-ala 84.2 1.22 ±0.16 169.8 ± 24   0.006 V04099X1 N = 16 *** 339385 D-ala-Phor18- 65.51.42 ± 0.08 Not lytic 0 LHRH N = 8 *** 339347 Phor18-Lupron 83.2 1.95 ±0.1  21 ± 1.2 0.09 N = 8 Significance compared to Phor21-βCG-ala(323033) *** p < 0.0005, ** p < 0.005, * p < 0.05

Acute hemolytic activities for 21 fusion constructs includingPhor18-Lupron and D-ala-Phor18-LHRH, were studied. The results aresummarized in FIGS. 5 and 6, and Table 3.

Hemolytic activity was determined in 96 well plates using a serialdilution of peptides exposed to 0.5% human RBC. Controls were saline (noRBC death) or 0.1% Triton X 100 (100% RBC lysis). The peptideconcentrations ranged from 0 to 100 μM. Incubations were conducted for 2h.

To ascertain the IC₅₀ of various fusion constructs on different humancancer cell lines compared to cisplatinum, IC₅₀ of fusion constructswere evaluated as in Table 3. The results are shown below:

IC₅₀ [μM] values in Human Cancer Cell Lines compared to CisplatinumD-ala- D-ala-Phor18- Phor18βCG- Cell Line Cisplatinum Phor18-LHRH LHRHPhor18-βCG ala MDA-MB- Not 0.86 ± 0.16 1.42 ± 0.08 1.35 ± 0.15  1.6 ±0.16 435S.luc determined MDA-MB- HCT 5.5 ± 1.2 33.7 ± 6.7  6.1 ± 0.620.5 ± 7.2  231 AN3-CA 11.85 ± 0.16   3.8 ± 0.08 40.6 ± 0.15 22.15 ±0.16  36.8 ± 0.16 OVCAR-3  184 ± 0.16   3 ± 0.5 13.8 ± 0.3  8.8 ± 0.411.6 ± 0.3  SKOV-3 321 ± 10  11.8 ± 0.3  19.2 ± 0.2  10.9 ± 0.6  18.9 ±0.4  LNCaP 19.9 ± 1.4  1.55 ± 0.08  5.0 ± 0.15 10.05 ± 0.16  15.5 ± 0.16Breast cancer cell lines: MDA-MB-435S.luc, MDA-MB-231 Ovarian cancercell lines OVCAR-3, SKOV-3 Prostate Cancer cell line: LNCaP Endometrialcancer cell line: AN3-CA

The data in general indicate very low hemolytic activities for thefusion constructs studied, except for Phor21-LHRH, LHRH-Phor21 andPhor18-Lupron. Under similar conditions the following fusion constructsdid not show any hemolytic activity (see FIG. 5): Phor15-aminocaproicacid-β-CG-ala (337474), D-ala-Phor21 β-CG-ala (337481)>150,000 μM,Phor21, unconjugated Phor 18=CLIP71, (KKKFAFA)₃ (SEQ. ID NO.16)-βCG-ala, Phor 21 (338982), Phor18=CLIP71 (338983), D-ala-Phor18-LHRH(339385) and (KKKFAFA)₃ (SEQ. ID NO. 16)-LHRH (338984). D-amino acidenatiomers had no measurable hemolytic activity.

Fusion constructs with hemolytic activities <50 μM were as follows:Phor21-LHRH (25 μM), LHRH-Phor21 (33 μM) and Phor18-Lupron (21 μM).

Fusion constructs with hemolytic activities >100 μM were as follows:Phor18-β-CG-ala (337476), and Phor18-LHRH (338613).

Fusion constructs with hemolytic activities between 50-100 μM were asfollows: (KKKFAFA)₃ (SEQ. ID NO. 16)-LHRH (95 μM), Phor21-βCG-ala andPhor21-ASAAS-βCG-ala had similar HA₅₀ of 70 μM.

Fusion constructs with hemolytic activities between 400-1300 μM were asfollows: Phor14-βCG-ala (337464), Phor18 GSGGS β-CG-ala (337467), Phor18ASAAS β-CG-ala, (337470), Phor15 ASAAS β-CG-ala (337471),D-ala-Phor21-LHRH (338611), and Phor18-ASAAS-LHRH (338612).

A clinically significant criterium is ratio of cell toxicity (IC₅₀) andHemolytic Activity (HA₅₀), or IC₅₀/HA₅₀ (FIG. 6, Table 3). In vivostudies were performed using a maximal concentration of 10 μM fusionconstruct which is several factors below the HA₅₀ values measured formost fusion constructs.

LHRH-conjugates with D-ala-Phor21, Phor18-ASAAS, D-ala-Phor18 and Phor18had very low IC₅₀/HA₅₀ ratios of 0.001-0.006 (compare Phor21-βCG-ala0.03). Toxicity to MDA-MB-435S.luc cells is significantly highercompared to Phor21-βCG-ala:D-ala) Phor21-LHRH is 3 times more toxic thanPhor21-βCG-ala, Phor18-LHRH and Phor18-ASAAS-LHRH is 2 times morepotent, D-ala-Phor18-LHRH is 1.5 more potent (FIG. 6).

In summary, fusion constructs evaluated by criteria of increasedtoxicity and less hemolytic activity (IC₅₀/HA₅₀ ratio) would be:Phor18-βCG-ala, Phor18-ASAAS-βCG-ala, Phor15-ASAAS-βCG-ala,Phor15-C6-βCG-ala, D-ala-Phor21-LHRH, Phor18-LHRH, Phor18-ASAAS-LHRH,and D-ala-Phor18-LHRH.

Example 3

This example describes in vivo studies in a mouse xenograft model ofbreast cancer with various types and doses of βCG- and LHRH-fusionconstructs.

Female Nu/Nu mice were injected subcutaneously with aMDA-MB-435S.luc/Matrigel HC suspension (1×10⁶ cells). The treatmentschedule is shown in FIG. 7. In brief, treatment started on day 13 aftertumor cell injection and continued on day 19 and 25. Treatments were:saline control, Phor21 (5 mg/kg), Phor18 (5 mg/kg), (KKKFAFA)₃ (SEQ. IDNO. 16) peptide-βCG-ala (5 mg/kg), Phor21-βCG-ala (0.01, 1 and 5 mg/kg),Phor18-βCG-ala (0.01, 1 and 5 mg/kg), D-ala-Phor21-βCG-ala (0.01, 1 and5 mg/kg), baseline 8-12 mice per group, 14 groups. The doses for weeklyinjections were 5, 1 and 0.01 mg/kg body weight, given as a bolus singleinjection.

All groups of mice tolerated the injections well. Only one mouse died ateach injection with 337476 at the 5 mg/kg dose. Death was an acuteevent. All mice in other treatment groups survived. No mice died as aconsequence of injection later than 10 minutes post injection.

The effect of cytolytic peptide injections on the primary tumors isillustrated in FIG. 8. In brief, the FIGS. 8A-8C show tumor volumeduring the course of the study for each individual peptide. FIGS. 8D-8Gshow tumor characteristics at necropsy: tumor volume (D), tumor weight(E), live tumor cells (F), tumor conditions (G).

Treatment efficacy was calculated as difference between measurements atthe beginning of treatment compared to the measurement at the end ofstudy (FIG. 8H-8I). FIG. 8J shows bodyweight of mice at necropsy.Viability of cells in the tumors was determined at the end of the studywhen tumors were measured for luciferase activity.

Tumor volume decreased significantly in all animals treated withpeptides containing βCG as a ligand (II, A), p<0.05 compared to baseline(except for 323033) at 0.01 mg/kg and the (KKKFAFA)₃ (SEQ. ID NO.16)-βCG-ala, Phor 21 and unconjugated Phor 18=CLIP71 controls. Tumorvolumes were significantly reduced compared to saline controls in alltreatment groups except for (KKKFAFA)₃ (SEQ. ID NO. 16)-βCG-ala, Phor 21and Phor 18, which were ineffective in reducing the xenograft volume.

Tumor weights also decreased significantly (p<0.001) in all treatmentgroups with βCG conjugated peptides when compared with animals treatedwith saline or (KKKFAFA)₃ (SEQ. ID NO. 16) peptides. Tumor cellviability, as measured by luciferase activity, correlated well withobserved changes in tumor weights and tumor volumes.

Treatment efficacy measured as reduction of tumor weight or viable tumorcells compared to baseline values showed a concentration dependenttreatment response for 323033 and 337476. 337481 showed consistentlyreduction of tumor load and live tumor cells at 0.01, 1 and 5 mg/kgdosages. 323033 was most effective at 1 mg/kg dose compared to 5 mg/kg(we have observed this in previous experiments already), but is onlysignificantly different to saline controls at the lowest dose applied.Fusion constructs 337476 and 337481 are significantly more effective at5 and 0.01 mg/kg compared to 323033 (p<0.0001) in reducing tumor loadsand viable tumor cells (p<0.004) below the baseline values. Fusionconstruct 337481 shows no concentration dependency and was the mosteffective of all tested constructs.

Cystic tumors were found in mice treated with fusion constructs 337476and 337481, which occurred to 80-90%, but only to 30% in the 1 mg/kg323033 treatment group. (Cyst formation has not been seen in thisxenograft model. The cysts consisted of liquid filled capsules.)Although it is not clear, it has been postulated that cystic tumorsoccur when cells are killed rapidly in a fast growing tumor. Cysts werepresent in prostate tumor xenografts treated with Phor 21.

Blood chemistry and complete blood count results for the treatmentgroups revealed that in no case did treatments affect liver, kidney,heart function. Platelet count, WBC and RBC counts were within normalrange, indicating that the treatment does specifically kill tumor cells,and did not cause anemia at the given concentration nor affected anyother observable vital body function. The fusion constructs were welltolerated with no long term side effects.

Based upon the foregoing in vivo tumor efficacy data, Phor18-βCG-ala(337476) and D-ala-Phor21-βCG-ala (337481) are significantly more potentthan reference Phor21-βCG-ala (323033) with respect to tumor weightreduction (p<0.0001) and destruction of viable tumor cells (p<0.004)compared to baseline values. Both fusion constructs did not causehemolysis in vivo and did not show persistent side effects at thehighest dose used (5 mg/kg). It is possible that tumor efficacy ofPhor18-βCG-ala would be greater with multiple injections even at thelowest dose.

For LHRH fusion constructs, the mouse xenograft model for breast cancerwas used. In brief, Nu/Nu female mice, outbred strain, age 5 weeks(Charles River) were injected subcutaneously with aMDA-MB-435S.luc/Matrigel suspension (1×10⁶ cells). Treatment was startedon day 21 after tumor cell injection and continued on day 26 and 29. Thedoses for weekly fusion construct injections were 2, 0.2 and 0.02 mg/kgbody weight, given as a bolus single injection. All mice were necropsied34 days after tumor cell injection-baseline values for tumor weightswere obtained by sacrificing 8 mice at treatment start. Primary tumors,liver, kidney, pancreas, heart, lung, and spleen were collected andprepared for histological evaluation in formalin. Tumor weights wererecorded at necropsy, part of the tumors were frozen at −80° C. forluciferase assay determination.

Treatment groups included saline control, Phor21-βCG-ala-323033 (0.02,0.2 and 2 mg/kg), D-ala-Phor21-LHRH-338611 (0.02, 0.2 and 2 mg/kg),(KKKFAFA)₃ (SEQ. ID NO. 16) LHRH-338614 (5 mg/kg), Phor18-LHRH-338613(0.02, 0.2 and 2 mg/kg), Phor18-ASAAS-LHRH-338612 (0.02, 0.2 and 2mg/kg), D-ala-Phor18-LHRH-339385 (0.02, 0.2 and 2 mg/kg), andPhor18-Lupron-339347 (0.02, 0.2 and 2 mg/kg), baseline 12 mice pergroup.

All groups tolerated the injections well. Only two mice died during thesecond and third injection with Phor18-ASAAS-LHRH at the 2 mg/kg dose(these mice were from the same cage). Death was an acute event. All micein other treatment groups survived. No mice died as a consequence ofinjection later than 10 minutes post injection.

FIG. 9 summarizes the effects of fusion construct injections on theprimary tumors as volume measures during the course of the study foreach individual construct. In all groups tumor volumes decreased duringtreatment except for mice treated with the (KKKFAFA)₃ (SEQ. ID NO.16)-LHRH conjugate or in saline control, where exponential tumor growthwas observed. The tumor volume recorded after 30 days post treatmentshows a reduction (p<0.01) compared to baseline for all fusionconstructs with the smallest tumor volumes recorded in treatment groupswith Phor18-ASAAS-LHRH and Phor18-LHRH.

Characteristics of tumors at necropsy are summarized in FIG. 10: (A)tumor weight, (B) changes of tumor weights compared to baseline, (C)total number of live tumor cells, (D) changes of total live tumor cellscompared to baseline, and (E) bodyweights at baseline and necropsy.Viability of tumor cells was determined at the end of the study whentumors were measured for luciferase activity. Treatment efficacy wascalculated as difference between measurements at the beginning oftreatment compared to the measurement at the end of study (FIGS. 10B and10D).

Tumor weights and total numbers of live tumor cells decreasedsignificantly in all animals in all treatment groups even at the lowestdose of 0.02 mg/kg when LHRH conjugates were given compared to salinecontrol and to (KKKFAFA)₃ (SEQ. ID NO. 16)-LHRH conjugated peptide(p<0.0001). Total tumor weights decreased compared to baselinesignificantly in all animals treated with 2 mg/kg peptides containingLHRH and at 0.02, 0.2 and 2 mg/kg for D-ala-Phor18-LHRH (A), (p<0.05).

The following concentrations resulted in tumor weights similar tobaseline: Phor21-βCG-ala-323033) at 0.02 mg/kg (p<0.07) and 0.2(p<0.06); Phor18-Lupron at 0.2 and 0.02 mg/kg dosage, Phor18-LHRH at 0.2mg/kg, and D-ala-Phor21-LHRH at 0.2 mg/kg. When total tumor weights werecompared to 0.02 mg/kg dose of Phor21-βCG-ala, Phor18-ASAAS-LHRH andD-ala Phor18-LHRH were superior at 0.02 mg/kg dosage (p<0.05).

The number of live tumor cells was determined and plotted in FIG. 10C astotal number of live tumor cells and in FIG. 10D as changes in livetumor cells compared to baseline. Cell viability, as measured byluciferase activity, correlated with the observed changes in tumorweight and tumor volume except for Phor18-ASAAS-LHRH and Phor18-LHRH,where a reduction of live tumor cells was observed. Treatment withD-ala-Phor18-LHRH (339385) and Phor18-ASAAS-LHRH (338612) were superiorto Phor21-βCG-ala at 2 mg/kg dose (p<0.04).

Treatment efficacy measured as a reduction of tumor weight or viabletumor cells compared to baseline values showed a concentration dependenttreatment response for all fusion constructs except forD-ala-Phor21-LHRH regarding tumor weights and live tumor cells.D-ala-Phor18-LHRH showed consistently reduction of tumor weights andlive tumor cells at 0.02, 2 and 2 mg/kg dosages. Most effective fusionconstructs in this experiment was Phor18-ASAAS-LHRH (338612), andD-ala-Phor18-LHRH (339385) in reducing the number of live tumor cellsand tumor weights at 2 mg/kg. Phor18-ASAAS-LHRH and D-ala-Phor18-LHRHwere superior compared to 2 and 0.02 mg/kg dose of Phor21-βCG-ala,(p<0.05). Phor18-Lupron was least effective at reducing tumor weight andlive tumor cells.

Blood chemistry and complete blood count results for the treatmentgroups revealed that in no case did the treatments affect liver, kidney,heart function. Platelet count, WBC and RBC counts were within normalrange, suggesting that the treatment does specifically destroys tumors,and did not cause anemia at the given concentration and they did notaffect any other observable vital body functions. Elevated potassiumlevels of 1.5 fold compared to saline controls were observed in miceinjected with Phor18-Lupron, Phor18-LHRH and D-ala-Phor21-LHRH. Thefusion constructs were well tolerated with no long term side effects.

Based upon the foregoing in vivo tumor efficacy data, Phor18-ASAAS-L HRH(338612), and D-ala-Phor18-LHRH (339385) are significantly more potentthan reference Phor21-βCG-ala with respect to tumor weight reduction(p<0.05) and destruction of viable tumor cells (p<0.04). Equallyeffective as Phor21-βCG-ala were D-ala-Phor21-LHRH, and Phor18-LHRH.Both fusion constructs did not cause hemolysis in vivo or other sideeffects. It is possible that the efficacy of Phor18-ASAAS-LHRH (338612),and D-ala-Phor18-LHRH (339385) would be greater with multiple injectionseven at the lowest dose.

Example 4

This example describes in vitro and in vivo receptor expression andspecificity studies.

LH receptor expression density both before and after treatment will beanalyzed to determine if treatment results in down regulation ofreceptor expression. Immunocytochemistry in comparison to Western Blotassays and RIA for quantification. LH and LHRH receptor determination inMDA-MB-435S.luc cells, CHO and TM4 cells using IHC with chamber slides,and Western Blot techniques. The same passage number of each cell linewill be tested for sensitivity to lytic peptide CG and lytic peptideLHRH.

Specificity of LH receptor fusion constructs was analyzed inMDA-MB-435S.luc (both LHRH and CG receptors), TM4 (no LHRH receptors)and CHO (no CG receptors) cells. IC₅₀ data show a significant reductionin sensitivity in TM4 cells for LHRH-Phor21 (10.9 μM), whereas CHO cellsshow low sensitivity to Phor21-βCG-ala (24.6 μM) when compared toMDA-MD-435S.luc cells (2.3 μM) (FIG. 12).

In vivo receptor expression is measured in connection with fusionconstruct treatment. In vivo receptor expression is measured atbeginning and end of fusion construct treatment.

Example 5

This example describes combination treatment with fusion constructs anda chemotherapeutic agent.

Preliminary studies of ovarian cancer cell lines (OVCAR-3 cells, multidrug resistant) incubated for 48 h with Phor21-βCG-ala and doxorubicinwere performed. Cell viability was determined as formazan reduction. Adecrease of IC₅₀ with increasing doxorubicin treatment in simultaneousincubation with Phor21-βCG-ala. The decrease is from 10 μM to 0.9 to 0.3to 0.05 μM at doxorubicin concentrations of 0, 0.5, 2 and 10 μg/ml.(FIG. 11). Treatment with Phor21-βCG-ala potentiated the response todoxorubicin. The data showed that Phor21-βCG-ala is more effective (13fold) when cells are co-incubated with doxorubicin.

Pretreatment of cancer cells with doxorubicin or cisplatinum, followedby treatment with LH or LHRH fusion constructs in the presence andabsence of LH or LHRH, will show if cytotoxicity of cells to theconstruct is altered.

In vivo efficacy of combination treatment was tested in a xenografttumor model (MDA-MB-435S). Mice are treated with a combination of afusion construct and a chemotherapeutic drug and compared to appropriatecontrols. Mice were treated according to the standard schedule used forthe drug, and are treated once a week for 3 weeks with the fusionconstruct.

The fusion conjugates have a high safety margin considering parameterssuch as hemolytic activity, maximum tolerated dose (MTD) (up to 16-25mg/kg) in comparison with the anti-tumor effective dose (0.02 or 0.01mg/kg). The safety margin for Phor21-βCG-ala is 16 whereas a value of800 can be reached for Phor18-βCG-ala and Phor18-LHRH. In comparison thesafety margin for Phor18-Lupron is only 8.

TABLE 6 below is a summary of the conjugates according to variouscriteria. In vivo Increased Perfor- in vitro Hemolytic mance Perfor-activity Activity IC₅₀/ compared MTD mance Peptide fold¹ [μM]² HA₅₀ to33⁴ mg/kg⁵ Rating Phor21- 1 73.4 0.03 1 16 βCG-ala (33) D-ala- 1.5 Not 0Superior 25 14 Phor18- lytic LHRH (85) Phor 18- 2.11 169 0.006 Superior16 13 βCG-ala (76) Phor18- 2.65 297 0.003 1 16 13 LHRH (13) D-ala- 1 Not0 Superior  8  11* Phor21- lytic βCG-ala (81) Phor18- 2.62 410 0.002Superior No data 11 ASAAS- LHRH (12) D-ala- 3.08 672 0.001 1 No data 10^(a) Phor21- LHRH (11) Phor18- 1 21 0.04 Less 16  5 Lupron (47) 711.6 421 0.003 No data No data 74 1.8 Not 0 No data No data lyticRating Code:Points allocated: 1 2 3In vitro activity 1×2×3×HA₅₀<50 50-100>100IC₅₀/HA₅₀<0.03<0.006<0.004In vivo efficacyCompared to 33 equal betterMTD comparedTo 33 equal better

1) IC₅₀ of 33 was 2.31 μM, values expressed as IC₅₀ of 33/IC₅₀ peptide.

2) Hemolytic activity expressed as HA₅₀ [μM].

3) In vivo performance refers to significance in tumor weight reductionand life tumor cell reduction vs baseline values compared to the sameparameters of peptide 33.

4) Injected dose resulting in 66.6% survival (acute and 8-14 days postinjection).

Peptide Codes:

33=Phor21βCG-ala

76=Phor18-βCG-ala

81=D ala Phor21-βCG-ala

85=D ala Phor18-LHRH

47=Phor18-Lupron

13=Phor18-LHRH

11=D ala Phor21-LHRH

12=Phor18-ASAAS-LHRH

71=Phor15-βCG-ala

74=Phor15-C6-βCG-ala

Example 6

This example describes peptide KFAKFAKKFAKFAKKFAKQHWSYGLRPG (SEQ. ID NO.15) (Phor18-LHRH (338613)) in vitro kinetic studies in various cancercell lines.

Standard chemotherapeutic drugs interact through DNA intercalation,microtubule interaction or are inhibitors of signal transductionpathways. Hence, their mechanism of action determines the time framenecessary to destroy target cells. The kinetics for doxorubicin in vitroto destroy human breast cancer cells such as MDA-MB-435S has beenreported to be as rapid as 4 hours, and other standard of caretreatments may even take longer. The most common mechanism of action indestroying cancer cells is apoptosis. As a reversible process and due tothe occurrence of multi-drug resistance (MDR), action of Pgp pumps thatexport drug molecules in MDR cancer cells, standard of care treatmentscan be ineffective.

In contrast to chemotherapeutic drugs, direct membrane action candestroy cancer cells within minutes. Membrane active compounds such ascationic lytic peptides include Phor18-LHRH (338613)(KFAKFAKKFAKFAKKFAKQHWSYGLRPG (SEQ. ID NO. 16)).

To determine the kinetics of cytotoxicity detailed time course studieswere conducted using Phor18-LHRH (338613) in comparison to theuntargeted lytic peptide moiety Phor 18=CLIP71 (338983) in various celllines expressing LHRH target receptors at various levels. Membranes ofnon-cancerous cell lines are neutral and are resistant to cytolyticpeptides, in contrast to cancer cell lines, which have a highphosphatidic acid content in their outer membrane.

The breast cancer cell lines studied were MDA-MB-435S (estrogen receptoralpha negative), MCF-7 and T47D (estrogen receptor alpha positive),ovarian cancer cell lines (OVCAR-3 and SKOV-3), prostate cancer (LNCaP),the non-malignant breast epithelial cell line MCF-10A and the mousefibroblast cell line NIH:3T3. The role of the LHRH receptor targeting inefficacy of Phor18-LHRH (338613) was also evaluated.

Cells were seeded into 96 well plates at a density of 10,000 cells/well.Treatment was initiated after 48 h by adding Phor18-LHRH (338613) (APC338613, Lot #P080401) or Phor18=CLIP71 (APC 338983, Lot #W08033C1) atconcentrations of 0.0001, 0.001, 0.01, 0.1, 1, 5, 10, 50 and 100 μM.Controls contained USP saline or 0.1% TritonX-100™ as reference for 0and 100% cell death, respectively. Incubations were terminated byremoving the culture media after 2 minutes, 5 minutes, 15 minutes, 30minutes, 1 hour, 2 hours, 6 hours, 24 and 48 hours. Cell viability wasassessed through formazan conversion assay (MTT assay, (CellTiter 96Aqueous One, Promega #G3582). Formazan conversion was determined using aBioRad Benchmark Plus microplate spectrophotometer dual wavelengths at490/630 nm at room temperature).

Data were calculated as fraction of absorption in TritonX-100™containing wells representing 100% cell death versus 0% cell death insaline containing wells. Cytotoxicity was determined as IC₅₀ usingGraphPad Prism version 5.01 for Windows, GraphPad Software, San DiegoCalif. USA according to the program for sigmoidal dose response withvariable slope using constraint values of 0 and 100%. 4 wells of eachindividual set of two 96 well plates were compared and analyzed.Statistical analysis for significance was determined by a two-tailedStudent's T-test.

In MDA-MB 435S cells (p250) Phor18-LHRH (338613) exhibited its maximalefficacy after 1 hour of incubation whereas CLIP71 incubations resultedin IC₅₀ values of 100, 109, 171, 145, 148, 86, 54.5, 55.1 (24 hours),and 3 [μM] (48 hours) (p<0.005) with increasing incubation time.Phor18-LHRH (338613) is a fast acting agent (1.2 μM 0.5 hour and 0.6 μMafter 1 hour) compared to the unconjugated lytic peptide CLIP71(>100μM).

In MCF-7 cells (p152) Phor18-LHRH (338613) exhibited its maximalefficacy after 1 hour of incubation whereas CLIP71 incubations resultedin IC₅₀ values of 92, 95, 50 and 22 [μM] (p<0.005) with increasingincubation time. Phor18-LHRH (338613) is a fast acting agent (3.4-1.8μM) compared to the unconjugated CLIP71.

In OVCAR-3 cells (p47) Phor18-LHRH (338613) exhibited its maximalefficacy after 1 hour of incubation whereas unconjugated Phor18=CLIP71(33 incubations resulted in IC₅₀ values of 337, 126, 85.5, 52.5, 22.9and 23.1 [μM] (p<0.005) with increasing incubation time. Phor18-LHRH(338613) is a fast acting agent with IC50 values of 6.7, 5.6, 5.3, 1.6,1.5, 0.5 and 0.5 [μM] (p<0.005) with increasing incubation time. Therapid kinetic data suggest that Phor18-LHRH (338613) and CLIP71 killcells by a different mechanism of action and increases the efficacy ofthe drug.

In SKOV-3 cells (p40) Phor 18-LHRH (338613) exhibited its maximalefficacy (11.5 μM) after 24 hours of incubation whereas CLIP71incubations resulted in IC₅₀ values of 86, 96, 53 and 50 [μM](p<0.005)with increasing incubation time. Phor18-LHRH (338613) is not anappropriate target for SKOV-3 cells since these cells do not presentfunctional LHRH receptors.

All cell lines presenting functional LHRH receptors such as breastcancer cells (MDA-MB-435 and MCF-7) and OVCAR-3 treated with Phor18-LHRH (338613) demonstrated the maximum effect (IC₅₀ in μM) within0.5-1 hour of incubation. In contrast, 24 hours incubation was requiredfor the maximal effect of CLIP71. Similar results were obtained for celllines that present functional LHRH receptors such as T47D, LNCaP. Incell lines that do not present functional LHRH receptors (SKOV-3 and HEK1A), Phor 18-LHRH (338613) and Clip71 showed similar toxicity with IC₅₀values of 0.10.3 resp 11.8 μM after 24 hour incubations. Non cancerouscell line 3T3 was highly resistant to Phor 18-LHRH (338613) and CLIP71with IC₅₀ values of >40 μM for CLIP-71 and >10 μM for Phor 18-LHRH(338613).

These results indicate that LHRH targeting enhances the efficacy ofCLIP71 and that non-cancerous cell lines are resistant to destruction bycytolytic cationic peptides. Phor18-LHRH (338613) shows remarkablepotency in destroying cancer cells through a receptor targeted mechanismwithin less than 1 hour. Phor18-LHRH (338613) is effective withinminutes and has advantages over standard chemotherapy that depend onintracellular uptake and interference with metabolic pathways andproliferation machinery for efficacy. Furthermore, Phor18-LHRH (338613)is capable of acting on multi-drug resistant cancer cells.

Example 7

This example includes data indicating a likely mechanism of action ofpeptide KFAKFAKKFAKFAKKFAKQHWSYGLRPG (SEQ. ID NO. 15) (Phor18-LHRH(338613)) on breast cancer cells in vitro.

To demonstrate a possible mechanism of action in vitro a fluorescencemicroscopic study was conducted in the human breast cancer cell lineMDA-MB-435 that over express LHRH receptors. In brief, human breastcancer cells (MDA-MB-435, passage #252) were seeded onto culture dishes.The following markers were introduced prior to adding EP100: DRAQ5™(Alexis Corporation)—blue—was used for staining of the nucleus andMitoTracker® Red CMXRos (M7512) (Molecular Probes, Inc. OR) were appliedfor visualizing intact mitochondria. Cell membranes were stained withwheat germ alexa fluor green conjugates (Molecular Probes, Inc. OR).

Cells were loaded first with Mitotracker dye, according to themanufacturers recommendation. Phor18-LHRH (338613) reconstituted insaline was added at a final concentration of 10 μM and incubated for5-10 minutes. Culture dishes with saline only served as controls. Thesupernatant was removed and the remaining cells prepared forfluorescence microscopy imaging.

A fluorescence microscopic evaluation of MDA-MB-435 presentingfunctional LHRH receptors breast cancer cells in vitro followingexposure to Phor18-LHRH (338613) revealed disintegration of the plasmamembrane after 5 minutes exposure to Phor18-LHRH (338613) (10 μM). Theseobservations suggested that Phor18-LHRH (338613) destroyed the plasmamembrane, leading to the death of the cell within minutes.

A fluorescence microscopic evaluation of SKOV-3 (p 41) and MDA-MB-435S(p 250) cells in cultures incubated for 30 minutes with 2 μM Phor18-LHRH(338613) FITC revealed that in SKOV-3 cells intracellular uptake wasabsent, membrane blebbing did not occur, and mitochondrial dye wasretained. In contrast in MDA-MB-435S cells intracellular uptake of Phor18-LHRH (338613) FITC was visible within 30 minutes as well as extensivemembrane blebbing leading to vesicle formation of the outer membrane andfading of mitochondrial dye. These observations suggest cell deathoccurred within minutes.

Within minutes, Phor18-LHRH (338613) destroyed cells that presentfunctional LHRH receptors. Phor18-LHRH (338613) destroyed cancer cellsthrough desintegrating the outer plasma membrane leading to necrosis.The mechanism of action strongly suggests a fast interaction ofPhor18-LHRH (338613) with the plasma membrane of cells that present thefunctional target. Cells that were negative for LHRH receptors were nota target and remained intact.

These data showed high specificity and efficacy of Phor 18-LHRH (338613)as an anticancer drug. Phor18-LHRH (338613) was effective within minutesand had major advantages over standard chemotherapy that requireintracellular uptake and interference with metabolic pathways andproliferation machinery for efficacy. Furthermore, Phor 18-LHRH (338613)was capable of acting on multi-drug resistant cancer cells.

Example 8

This example includes studies demonstrating that peptideKFAKFAKKFAKFAKKFAKQHWSYGLRPG (SEQ. ID NO. 15) (Phor18-LHRH (338613)) waseffective against cancer in a xenograft models.

In vivo efficacy studies were conducted as monotherapy, or a combinationtherapy with standard of care treatments in nude mice bearing humanbreast cancer xenografts: MDA-MB-435S.luc (s.c.), MCF-7 (estrogenreceptor alpha positive), human ovarian cancer xenografts: OVCAR-3,(s.c.), human prostate cancer xenografts: PC-3 (androgen receptornegative). Phor18-LHRH (338613) dissolved in saline (0.02, 0.2 and 2mg/kg) was injected once or twice a week for 3 weeks as a single bolusinjection in into the lateral tail vein.

PHOR 18-LHRH (338613) Combination therapies were conducted in a MCF-7breast cancer xenograft models.

Mice were sacrificed one week after the last injection and bloodcollected for chemistry panel, tumor weights and body weights wererecorded. Part of the tumors were fixed in PBS buffered 10% formalin forhistological evaluation. The various in vivo xenograft studies aresummarized in Table 7.

TABLE 7 Xenograft Studies Treat- Median Median ment Tumor Tumor RegimenWeight vs Weight Xenograft and Baseline vs Saline Model Duration p <0.05 P < 0.05 Remarks OVCAR-3 1 × 3 wk, 0.2 mg/kg 0.2, 2 mg/kg Necrosis20 d in treated tumors, reduction of LHRH receptors MDA-MB- 1 × 3 wk,0.02, 2 mg/kg 0.02, 0.2 and 435S.luc 22 d 2 mg/kg MDA-MB- l × 3 wk,0.002 mg/kg 0.0002 mg/kg Necrosis 435S.luc 22 d and reduction of LHRHreceptors PC-3 1 × 3 wk, 0.2 0.002, 0.02, 21 d 0.2 mg/kg MCF-7 2 × 3 wk,0.02 mg/kg Necrosis 19 d growth delay in treated tumors 0.02 and 0.2

To determine the minimal effective dose necessary to reduce MDA-MB-435Sxenograft weights in a single dose regimen, Nu/Nu female mice, outbredstrain, age 5 weeks (Charles River), were injected (s.c.) into theinterscapular region with a MDA-MB-435S (passage #249)/Matrigelsuspension (2.4×10⁶ cells/mouse) [Leuschner 2006]. Phor18-LHRH (338613)(ID: 338613 lot#P080401) (0.00002, 0.0002, 0.002, 0.02, 0.2 and 1 mg/kg)and unconjugated Phor18=CLIP71 (APC 338983, Lot #W08033C1) plus LHRH(0.2/0.122 mg/kg, ([D-Trp6]-LHRH; Sigma L9761, lot #037K1103) werereconstituted in USP saline prior to dosing. The doses were given as asingle bolus intravenous injection via lateral tail vein once per weekfor 3 weeks.

Each group consisted of 16 mice, which were injected once a week for 3weeks. A group of 16 mice was sacrificed at the time of treatment startto serve as baseline. Saline injections served as control groups. Duringthe entire study tumor volumes were recorded twice weekly.

Treatment started on day 16 after tumor cell injection when the tumorswere established and continued on days 23 and 30. All remaining micewere necropsied 37 days after tumor cell injection.

Treatment response was determined by tumor weights and tumor weightchange at necropsy compared to saline controls and untargeted CLIP71treatment. Primary tumors were collected, weighed and prepared forhistological evaluation in formalin fixation with 10% PBS bufferedformalin. Statistical evaluation of data sets were conducted in GraphPadPrizm 4 and significance calculated by Wilcoxon signed-rank test.

Tumor volumes and tumor weights increased in saline controls and micetreated with clip71 plus LHRH. Tumor volumes and tumor weights werereduced significantly compared to saline controls in mice treated with0.0002 mg/kg Phor18-LHRH (338613). Treatment with doses of Phor18-LHRH(338613) as low as 0.002 mg/kg significantly reduced tumor volume andtumor weight compared to baseline (p<0.0002).

Histological evaluation of tumor sections from MDA-MB-435S xenograftedmice stained with hematoxylin/eosin from treated mice show viable tumorcells in saline control and mice treated with CLIP71/LHRH. In contrast,significant necrosis was evident in tumors from mice treated withPhor18-LHRH (338613) at doses as low as 0.0002 mg/kg. Untargetedcationic lytic peptide CLIP71 did not decrease tumor weights or destroytumor tissue.

Phor18-LHRH (338613) was highly effective in reducing tumor weights ofMDA-MB-435S xenografts as low as 0.002 mg/kg leading to necrosis intreated tumor tissues. Untargeted treatment with cytolytic peptides isineffective.

In order to determine the minimal effective dose necessary to reduceMDA-MB-435S xenograft weights in a multiple dose regimen, Nu/Nu femalemice, outbred strain, age 8 weeks (Charles River), were injected (s.c.)into the interscapular region with a MDA-MB-435S (passage #253)/Matrigelsuspension (2×10⁶ cells/mouse) as previously described above.Phor18-LHRH (338613) (ID: 338613 lot#P080401) (0.002, and 0.2 mg/kg)were reconstituted in USP saline prior to dosing. The doses were givenas a single bolus intravenous injection via lateral tail vein on days15, 16, 17, 20, 21, 22, 23, 27, 28, 29, 30, 33, 34, 35, 36, 37, 38, 40,41, 42 after tumor cell inoculation. Saline injections served as controlgroups.

Each treatment group consisted of 16 mice. During the entire study tumorvolumes were recorded twice weekly. Final necropsy was conducted on day45 after tumor cell injection. Injections were resumed due to occlusionof the tail vein in most mice. At study endpoint body weight, tumorweights were determined and fixed in phosphate buffered 10% formalin.

Treatment with hor18-LHRH (338613) using multiple intravenous injectionsresulted in tumor regression at both dose levels. Tumor free mice wereobserved in both treatment groups as 6/23 in group receiving 0.002 mg/kgand 1/20 at 0.2 mg/kg. Residual masses typically consisted of Matrigel.One mouse did not respond to treatment in group 0.2 mg/kg.

No necrosis in the tails was observed, no reddening of the tails waspresent. Bodyweights were not affected by treatment over the entirestudy period.

Survival was 100% in treated mice. In contrast, 8 mice in the salinecontrol group were sacrificed on day 30 post tumor cell injection (priorto study endpoint) because the tumor volume exceeded 2,500 mm³.

Histological examination of H&E stained tumors from mice treated with0.002 and 0.2 mg/kg Phor18-LHRH (338613) in multiple injection regimenshowed eradication of tumor cells in treated mice. In contrast, viabletumor cells were present in saline control mice.

Phor18-LHRH (338613) destroyed and reduced tumor weights significantlyand extended the lifespan of treated mice. The treatments were withoutany visible effects on body weight or organ examination.

Example 9

This example includes a description of peptideKFAKFAKKFAKFAKKFAKQHWSYGLRPG (SEQ. ID NO. 15) (Phor18-LHRH (338613))efficacy studies in a breast, ovarian and prostate cancer xenograftmodels.

In vitro studies described herein demonstrated that Phor18-LHRH (338613)is a fast acting agent, killing cancer cells within minutes of contact.To determine the efficacy of Phor18-LHRH (338613) on breast cancerxenografts in the initial phase of targeted treatment, kinetics of celldestruction through Phor 18-LHRH (338613) in a breast cancer xenograftmodel after a single injection of Phor18-LHRH (338613) was studied

In brief, Nu/Nu female mice, outbred strain, age 5 weeks (CharlesRiver), were injected (s.c.) into the interscapular region with aMDA-MB-435S (passage #249)/Matrigel suspension (2.4×10⁶ cells/mouse) asdescribed in Example 10. Phor18-LHRH (338613) (ID: 338613 lot#P080401)(0.2, and 2 mg/kg) were reconstituted in USP saline prior to dosing. Thedoses were given as a single bolus intravenous injection via lateraltail vein.

Mice were sacrificed 1, 2 and 16 hours after treatment with Phor 18-LHRH(338613) or saline. At study endpoint body weight, tumor weights weredetermined and tumors were fixed in phosphate buffered 10% formalin.

Histological evaluation from H&E stained sections from tumors showedviable tumor cells with multiple mitotic figures in saline treated mice.Treatment with Phor18-LHRH (338613) at both 0.2 and 2 mg/kg doses showeddestruction of tumors from MDA-MB-435S xenografts as rapidly as 1 hourafter injection.

Phor18-LHRH (338613) destroyed tumors as early as 1 h after dosing,suggesting a fast acting mechanism that causes cell death throughnecrosis. These data confirm that Phor18-LHRH (338613) acts through itsmembrane contact to LHRH receptor presenting tumor cells.

To determine the efficacy of Phor18-LHRH (338613) on ovarian cancerxenografts that resemble the human disease, single and multiple dosestudies were conducted. The xenograft model of the OVCAR-3 human ovariancancer cell line that present functional LHRH receptors was used in thisstudy. OVCAR-3 represents a slow growing xenograft model and secretesthe tumor marker (cancer antigen 125, or CA125). It's secretion can beused as treatment response and is a measure of drug activity.

The purpose of this xenograft study was to test Phor 18-LHRH (338613) ina ovarian cancer model, to determine if Phor18-LHRH (338613) iseffective in vivo in multi-drug resistant, slow growing tumor models assingle weekly injections. In brief, Nu/Nu female mice, inbred strain,age 5 weeks (Harlan-Sprague Dawley) were injected subcutaneously with aNIH:OVCAR-3 cells/Matrigel suspension (4.6×10⁶ cells/mouse). Treatmentwas started on day 33 after tumor cell injection when the tumors wereestablished and continued on days 41 and 47. The doses for the 3 weeklyinjections were 0.02, 0.2 and 2 mg/kg body weight, given as a singlebolus intravenous injection via lateral tail vein administered once aweek for three weeks on days 33, 41 and 47. Necropsies were conducted onday 52. Data are presented as mean SEM. Arrows show dosing.

Treatment groups included saline control (N=10), Phor18-LHRH (338613)(338613, V09108X1) (0.02 (N=10), 0.2 (N=10), and 2 mg/kg (N=9),), andunconjugated Phor18=CLIP71 (APC 338983, Lot #V04004X1) (2 mg/kg (N=10)),Cisplatinum/CP in saline (Calbiochem, Cat 232120, D0005495,) (10 mg/kg,3qd (N=10),), baseline (N=9).

A group of 9 tumor bearing mice was sacrificed on day 33 and served asbaseline group. All mice were necropsied 51-52 days after tumor cellinjection. Tumor volumes and bodyweights were recorded twice weeklyduring the study, as well as overall veterinarian examination of micewas conducted.

Primary tumors, liver, kidney, pancreas, heart, lung, and spleen werecollected, fixed in formalin and prepared for histological evaluation.Tumor weights were measured at necropsy, part of the tumors were frozenat −80° C. for LH/CG and LHRH receptor assay determination.

As a biomarker for drug activity, CA125 was determined in serum,collected from each individual mouse at necropsy using a Enzyme LinkedImmunoassay for quantitative determination of ovarian cancer antigenCA125 (Assay kit Genway, Biotech, Inc. San Diego, Calif., Catalog#40-052-115009, #BC-1013 according to the manufacturer).

LHRH receptor levels were assessed from formalin fixed tumors.Quantitative immunoperoxidase image analysis was conducted with theVentana Image Analysis System (VIAS) adjunctive computer assisted imageanalysis system functionally connected to an interactive microscope(Axio Imager). The quantitative analysis was conducted with the programfor quantification of Her2/neu receptor that included morphometric andcolorimetric analysis. Receptor status results were reported aspercentage of cells showing positive staining of the LHRH receptorsunder the following criteria: 0 non-immunoreactive, 1+: 1-25% positive,2+: 26-50% positive, 3+: 51-75% positive cells.

All mice groups tolerated the injections well. One mouse died during thefirst injection with Phor 18-LHRH (338613) at the 2 mg/kg dose. Deathwas an acute event and was procedural and not related to treatment. Allmice in other treatment groups survived. No mice died as a consequenceof injection later than 10 minutes after injection.

Tumor volumes decreased during treatment with Phor 18-LHRH (338613). Incontrast, for mice treated with the CLIP71, cisplatinum or in salinecontrols, tumor growth was observed. The tumor volumes recorded after 42days after tumor cell injection showed reductions (p<0.001) compared tobaseline at concentrations of Phor18-LHRH (338613) as low as 0.2 mg/kgbodyweight.

Characteristics of tumors at necropsy (median tumor weights and changesof median tumor weights compared to baseline) were determined. Reducedtumor weights compared to saline controls and unconjugated Phor18=CLIP71 (p<0.05) were obtained in the groups for 2 and 0.2 mg/kgdosages of Phor18-LHRH (338613). Tumor free mice were found in groups0.2 and 2 mg/kg of Phor 18-LHRH (338613). Treatment response measured astumor regression compared to treatment start was greatest in micetreated with Phor18-LHRH (338613) at 0.2 mg/kg (p<0.03 vs baseline).Cisplatinum and unconjugated Phor18=CLIP71 were not effective inreducing tumor weights.

Saline controls, CLIP71 and cisplatinum treated mice showed steady tumorgrowth. Serum levels for CA125 corresponded to tumor weights (r²=0.66).CA125 secretion was reduced in Phor18-LHRH (338613) treated mice andcompared to saline controls was greatest in mice treated withPhor18-LHRH (338613) at 0.2 and 2 mg/kg (p<0.0002).

Sizes of tumor excised from OVCAR-3 xenograft bearing mice aftertreatment with 0.02 mg/kg Phor18-LHRH (338613) were reduced and necroticcompared to saline controls. Treated tumors showed a reduction of LHRHreceptor levels by 1-2 score points. Tumor sections stained withhematoxylin/eosin showed significant necrosis in groups treated with0.02 mg/kg Phor18-LHRH (338613). Xenograft bearing mice treated withCisplatinum or CLIP71 has no reduction in tumor volume, LHRH receptorlevels and showed viable tumor cells after histological evaluation.

Treatment with Phor18-LHRH (338613) caused tumor regression, reductionof CA125 tumor marker in plasma, reduction of LHRH receptor levels andnecrosis in ovarian xenograft model. Phor18-LHRH (338613) is thereforeeffective in destroying multi-drug resistant ovarian cancer xenografts.

To determine the efficacy of Phor 18-LHRH (338613) on prostate cancerxenografts, the effect of Phor18-LHRH (338613) in vivo in a fastaggressive growing xenograft model was studied. PC-3 xenograftsuntreated cause significant weight loss in mice.

In brief, Nu/Nu male mice, outbred strain, age 6 weeks (Charles River)were injected subcutaneously with a PC-3 cells/Matrigel suspension(1×10⁶ cells/mouse). Treatment was started on day 15 after tumor cellinjection when the tumors were established and continued on days 22 and29. The doses for the 3 weekly injections were 2, 0.2 and 0.02 mg/kgbody weight, given as a bolus single intravenous injection via lateraltail vein. Treatment groups included saline control (N=12), Phor18-LHRH(338613) (APC 338613 Lot #V09108X1) (0.002 (N=12), 0.02 (N=12), 0.2(N=12) and 2 mg/kg (N=12),), and unconjugated Phor18=CLIP71 (338983, Lot#V04004X1) (5 mg/kg (N=12), baseline (N=12).

A group of 12 tumor bearing mice was sacrificed on day 15 and served asbaseline group. All mice were necropsied 35 and 36 days after tumor cellinjection. Tumor volumes and bodyweights were recorded twice weeklyduring the study, as well as overall veterinarian examination of micewas conducted.

Primary tumors, liver, kidney, pancreas, heart, lung, and spleen werecollected, fixed in formalin and prepared for histological evaluation.Tumor weights were measured at necropsy, part of the tumors were frozenat −80° C. for LH/CG and LHRH receptor assay determination.

All groups tolerated the injections well and all mice in treatmentgroups survived. No mice died as a consequence of injection.

Tumor volumes decreased during treatment with PHor18-LHRH (338613) atdoses of 0.002, 0.02, 0.2 and 2 mg/kg. For mice treated with the CLIP71or in saline controls, tumor growth was observed. The tumor volumesrecorded after 22 days after tumor cell injection showed reductions(p<0.001) compared to saline controls and CLIP71 at concentrations ofPhor18-LHRH (338613) as low as 0.002 mg/kg bodyweight. Tumor weightswere significantly reduced compared to saline controls and CLIP-71treatment groups (0.001 in all Phor 18-LHRH (338613) treated groups).

PC-3 xenografts are known to cause weight loss in nude mice. InPhor18-LHRH (338613) treated mice, tumor volumes decreased in groupstreated with 0.002, 0.02, 0.2 and 2 mg/kg Phor18-LHRH (338613) comparedto saline controls and CLIP71 injectections. Median tumor weights atnecropsy were significantly reduced compared to saline controls andCLIP71 (p<0.001). Mice in control groups were cachectic and suffered aweight loss of more than 10 g compared to treated mice.

Phor18-LHRH (338613) is effective in arresting tumor growth in PC-3xenografts and preventing severe weight loss due to tumor burden.Unconjugated Phor18-LHRH (338613) is ineffective.

In sum, the foregoing studies indicate that peptideKFAKFAKKFAKFAKKFAKQHWSYGLRPG (SEQ. ID NO. 15) (Phor18-LHRH (338613)) iseffective in vivo in destroying breast cancer, ovarian cancer andprostate cancer xenografts. Phor 18-LHRH (338613) causes tumor necrosisin treated mice, with necrosis being evident as early as 1 hour postinjection. Phor 18-LHRH (338613) is effective as a single weeklytreatment regimen inducing necrosis and causing tumor weight reduction.As a multiple weekly regimen eradication of tumors is more evidentincluding destruction of residual tumor cells. Phor 18-LHRH (338613)causes reduction in LHRH receptor levels after treatment, consistentwith target cell destruction.

What is claimed:
 1. A fusion construct comprising a first lytic domainand a second domain, wherein said first lytic domain consists of peptideKFAKFAKKFAKFAKK (SEQ. ID NO. 1) or peptide KFAKFAKKFAKFAKKFAK (SEQ. IDNO. 4); and said second domain comprises a luteinizing hormone releasinghormone (LHRH), an LHRH fragment, or an LHRH analog.
 2. The fusionconstruct of claim 1, wherein said LHRH comprises a sequence QHWSYGLRPG(SEQ. ID NO. 9).
 3. The fusion construct of claim 1, wherein said firstlytic domain is positioned at the NH₂-terminus relative to said seconddomain.
 4. The fusion construct of claim 1, wherein said second domainis positioned at the NH₂-terminus relative to said first lytic domain.5. The fusion construct of claim 1, wherein said first lytic domain orsaid second domain has one or more D-amino acids.
 6. The fusionconstruct of claim 1, wherein said first lytic domain has a D-amino acidat any K, F or A residue.
 7. The fusion construct of claim 1, whereinsaid first lytic domain forms an amphipathic alpha-helix.
 8. The fusionconstruct of claim 1, wherein said first lytic and second domains arejoined by a covalent bond.
 9. The fusion construct of claim 1, whereinsaid first lytic and second domains are joined by a peptide or anon-peptide linker.
 10. The fusion construct of claim 9, wherein saidnon-peptide linker comprises a linear carbon chain linker.
 11. Thefusion construct of claim 9, wherein said non-peptide linker comprises alinear 6 carbon chain linker.
 12. The fusion construct of claim 1,wherein said peptide linker has a length from 1 to 25 amino acidresidues.
 13. The fusion construct of claim 1, wherein said first lyticand second domains are joined by peptide linker comprising one or moreA, S or G amino acid residues.
 14. The fusion construct of claim 1,wherein said first lytic and second domains are joined by peptide linkercomprising or consisting of: GSGGS (SEQ. ID NO. 10), or ASAAS (SEQ. IDNO. 11).
 15. The fusion construct of claim 1, wherein said fusionconstruct is isolated or purified.
 16. The fusion construct of claim 1,wherein said fusion construct has greater anti-cell proliferativeactivity than Phor21-βCG-ala, Phor21-(SEQ. ID NO. 10)-βCG-ala,Phor21-(SEQ. ID NO. 11)-βCG-ala, or Phor 14-βCG-ala, as ascertained by alower IC₅₀ value.
 17. The fusion construct of claim 1, wherein saidfusion construct has a smaller IC₅₀/HA₅₀ (hemolytic activity) ratio,than Phor21-βCG-ala, Phor21-(SEQ. ID NO. 10)-βCG-ala, Phor21-(SEQ. IDNO. 11)-βCG-ala, or Phor 14-βCG-ala.
 18. The fusion construct of claim1, wherein said fusion construct has an IC₅₀/HA₅₀ (hemolytic activity)ratio of less than about 0.02, 0.01, or 0.005.
 19. The fusion constructof claim 1, wherein said first lytic domain consists of peptideKFAKFAKKFAKFAKK (SEQ. ID NO. 1).
 20. The fusion construct of claim 1,wherein said first lytic domain consists of peptide KFAKFAKKFAKFAKKFAK(SEQ. ID NO. 4).
 21. The fusion construct of claim 1 wherein the LHRHanalog comprises Lupron (leuprolide).
 22. The fusion construct of claim1, wherein the LHRH analog comprises zoladex (goserelin).
 23. The fusionconstruct of claim 1, wherein the LHRH analog comprises supprelin(histrelin).
 24. The fusion construct of claim 1, wherein the LHRHanalog comprises triptorelin.
 25. The fusion construct of claim 1,wherein the LHRH analog comprises buserelin.
 26. The fusion construct ofclaim 1, wherein the LHRH analog comprises centrorelix.
 27. The fusionconstruct of claim 3, wherein the LHRH analog comprises ganirelix. 28.The fusion construct of claim 1, wherein the LHRH analog comprisesabarelix.
 29. The fusion construct of claim 1, wherein the LHRH analogcomprises antide.
 30. The fusion construct of claim 1, wherein the LHRHanalog comprises teverelix.
 31. The fusion construct of claim 1, whereinthe LHRH analog comprises degarelix (Fe200486).
 32. A compositioncomprising the fusion construct of claim
 1. 33. A pharmaceuticalcomposition comprising the fusion construct of claim
 1. 34. Acomposition, comprising the fusion construct of claim 1 and analkylating agent, anti-metabolite, plant extract, plant alkaloid,nitrosourea, hormone, nucleoside or a nucleotide analog.
 35. Acomposition, comprising the fusion construct of claim 1 andcyclophosphamide, azathioprine, cyclosporin A, prednisolone, melphalan,chlorambucil, mechlorethamine, busulphan, methotrexate,6-mercaptopurine, thioguanine, 5-fluorouracil, cytosine arabinoside,AZT, 5-azacytidine (5-AZC), bleomycin, actinomycin D, mithramycin,mitomycin C, carmustine, lomustine, semustine, streptozotocin,hydroxyurea, cisplatin, carboplatin, oxiplatin, mitotane, procarbazine,dacarbazine, taxol (paclitaxel), vinblastine, vincristine, doxorubicin,dibromomannitol, irinotecan, topotecan, etoposide, teniposide,gemcitabine, or pemetrexed.